bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Residues in the Anticodon Recognition Domain of the Aspartyl-tRNA Synthetase from
Pseudomonas aeruginosa
Are Individually Implicated in the Recognition of tRNA
Asn
Authors: Bernard Dominic , Akochy Pierre-Marie , Beaulieu David , Lapointe Jacques , Roy Paul H. 
Published: December 2005

ABSTRACT

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA
Asn
but by specific tRNA-dependent transamidation of aspartyl-tRNA
Asn
. This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA
Asp
and tRNA
Asn
with aspartic acid. Recently, it has been shown for the first time in an organism (
Pseudomonas aeruginosa
PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA
Asn
but does not participate in Gln-tRNA
Gln
formation.
P. aeruginosa
PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA
Asn
. Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of
P. aeruginosa
AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA
Asp
charging over that of tRNA
Asn
by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA
Asp
and tRNA
Asn
. Therefore, these two determinants of specificity of
P. aeruginosa
AspRS could be important for all bacterial AspRSs.
Polymorphisms in the Nine Polymorphic Membrane Proteins ofChlamydia trachomatisacross All Serovars: Evidence for Serovar Da Recombination and Correlation with Tissue Tropism
Authors: Gomes João P. , Nunes Alexandra , Bruno William J. , Borrego Maria J. , Florindo Carlos , Dean Deborah 
Published: December 2005

ABSTRACTChlamydia trachomatisis an intracellular bacterium responsible for ocular, respiratory, and sexually transmitted diseases. The genome contains a nine-member polymorphic membrane protein (Pmp) family unique to members of the orderChlamydiales. Genomic and molecular analyses were performed for the entirepmpgene family for the 18 reference serological variants (serovars) and genovariant Ja to identify specific gene and protein regions that differentiate chlamydial disease groups. The mean genetic distance among all serovars varied from 0.1% forpmpAto 7.0% forpmpF. Lymphogranuloma venereum (LGV) serovars were the most closely related for thepmpgenes and were also the most divergent, compared to ocular and non-LGV urogenital disease groups. Phylogenetic reconstructions showed that for six of ninepmpgenes (notpmpA,pmpD, orpmpE), the serovars clustered based on tissue tropism. The most globally successful serovars, E and F, clustered distantly from the urogenital group for fivepmpgenes. Thesepmpgenes may confer a biologic advantage that may facilitate infection and transmission for E and F. Surprisingly, serovar Da clustered with the ocular group frompmpEtopmpI, which are located together in the chromosome, providing statistically significant evidence for intergenomic recombination and acquisition of a genetic composition that could hypothetically expand the host cell range of serovar Da. We also identified distinct domains forpmpE,pmpF, andpmpHwhere substitutions were concentrated and associated with a specific disease group. Thus, our data suggest a possible structural or functional role that may vary amongpmpgenes in promoting antigenic polymorphisms and/or diverse adhesions-receptors that may be involved in immune evasion and differential tissue tropism.
Cooperativity Between Different Nutrient Receptors in Germination of Spores of
Bacillus subtilis
and Reduction of This Cooperativity by Alterations in the GerB Receptor
Authors: Atluri Swaroopa , Ragkousi Katerina , Cortezzo Donna E. , Setlow Peter 
Published: December 2005

ABSTRACT

The GerA nutrient receptor alone triggers germination of
Bacillus subtilis
spores with
l
-alanine or
l
-valine, and these germinations were stimulated by glucose and K
+
plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K
+
(GFK) (termed cogerminants) plus GerK for triggering of germination with a number of
l
-amino acids. GerB and GerA also triggered spore germination cooperatively with
l
-asparagine, fructose, and K
+
and either
l
-alanine or
l
-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to
d
-alanine do not respond to
d
-alanine but respond to the same
l
-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these
l
-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to
l
-alanine germination via GerA, spore germination via
l
-alanine and GerB or GerB* was not inhibited by
d
-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K
+
somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to
l
-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to
l
-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to
l
-amino acids, and GerA interacts only with
l
-valine,
l
-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to
l
-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.
Disruption of
sucA
, Which Encodes the E1 Subunit of α-Ketoglutarate Dehydrogenase, Affects the Survival of
Nitrosomonas europaea
in Stationary Phase
Authors: Hommes Norman G. , Kurth Elizabeth G. , Sayavedra-Soto Luis A. , Arp Daniel J. 
Published: December 2005

ABSTRACT

Although
Nitrosomonas europaea
lacks measurable α-ketoglutarate dehydrogenase activity, the recent completion of the genome sequence revealed the presence of the genes encoding the enzyme. A knockout mutation was created in the
sucA
gene encoding the E1 subunit. Compared to wild-type cells, the mutant strain showed an accelerated loss of ammonia monooxygenase and hydroxylamine oxidoreductase activities upon entering stationary phase. In addition, unlike wild-type cells, the mutant strain showed a marked lag in the ability to resume growth in response to pH adjustments in late stationary phase.
Role of the Novel OprD Family of Porins in Nutrient Uptake in
Pseudomonas aeruginosa
Authors: Tamber Sandeep , Ochs Martina M. , Hancock Robert E. W. 
Published: December 2005

ABSTRACT

To circumvent the permeability barrier of its outer membrane,
Pseudomonas aeruginosa
has evolved a series of specific porins. These channels have binding sites for related classes of molecules that facilitate uptake under nutrient-limited conditions. Here, we report on the identification of a 19-member family of porins similar to the basic-amino-acid-specific porin OprD. The members of this family fell into one of two phylogenetically distinct clusters, one bearing high similarity to OprD and the other bearing most similarity to the putative phenylacetic acid uptake porin PhaK of
Pseudomonas putida
. Analysis of the genome context, operon arrangement, and regulation of the PhaK-like porin OpdK indicated that it might be involved in vanillate uptake. This result was confirmed by demonstrating that an
opdK
mutant had a deficiency in the ability to grow on vanillate as a carbon source. To extrapolate these data to other paralogues within this family, the substrate specificities of 6 of the 17 remaining OprD homologues were inferred using an approach similar to that used with
opdK
. The specificities determined were as follows: OpdP, glycine-glutamate; OpdC, histidine; OpdB, proline; OpdT, tyrosine; OpdH,
cis
-aconitate; and OpdO, pyroglutamate. Thus, members of the OprD subfamily took up amino acids and related molecules, and those characterized members most similar to PhaK were responsible for the uptake of a diverse array of organic acids. These results imply that there is a functional basis for the phylogenetic clustering of these proteins and provide a framework for studying OprD homologues in other organisms.
Transcription of Virulence Factors in
Staphylococcus aureus
Small-Colony Variants Isolated from Cystic Fibrosis Patients Is Influenced by SigB
Authors: Moisan Hélène , Brouillette Eric , Jacob Christian Lebeau , Langlois-Bégin Philippe , Michaud Sophie , Malouin François 
Published: December 2005

ABSTRACT

Staphylococcus aureus
small-colony variants (SCVs) are believed to account in part for the persistence of
S. aureus
during chronic infections. Little is understood about the gene expression profile that may explain the phenotype and distinguish SCVs from prototype
S. aureus
strains. In this study, DNA array transcriptional profiles of clinical SCVs isolated from the airways of cystic fibrosis patients were obtained and compared to those obtained from a laboratory-derived SCV strain (i.e., a respiratory-deficient
hemB
mutant) and prototype
S. aureus
strains. The genes commonly up-regulated in both
hemB
and clinical SCVs were found to be implicated in fermentation and glycolysis pathways. The well-known virulence regulator
agr
was not activated in SCVs, and such strains had low levels of alpha-toxin (
hla
) gene expression. Clinical SCVs also had a transcriptional signature of their own. Of striking interest is that many genes, most of them under the positive control of the alternate sigma factor SigB, were specifically up-regulated and differed in that way from that seen in prototype
S. aureus
and the
hemB
mutant. Since SigB influences up-regulation of adhesin type genes while indirectly down-regulating exoproteins and toxins, we evaluated the internalization and persistence of SCVs in mammalian cells. Results showed that clinical SCVs persisted much more efficiently in cells than the
hemB
and prototype strains and that a
sigB
mutant was a poor persister. Thus, it appears that the
agr
locus plays a minor role in the regulation of the virulon of SCVs, unlike SigB, which may have a key role in intracellular persistence.
Soluble V Domain of Nectin-1/HveC Enables Entry of Herpes Simplex Virus Type 1 (HSV-1) into HSV-Resistant Cells by Binding to Viral Glycoprotein D
Authors: Kwon Heechung , Bai Qing , Baek Hyun-Jung , Felmet Kelly , Burton Edward A. , Goins William F. , Cohen Justus B. , Glorioso Joseph C. 
Published: December 2005

ABSTRACT

Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1
123
), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1
123
, approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1
123
did not associate with the cell surface. sNec1
123
-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.
Characterization of Early Steps in the Poliovirus Infection Process: Receptor-Decorated Liposomes Induce Conversion of the Virus to Membrane-Anchored Entry-Intermediate Particles
Authors: Tuthill Tobias J. , Bubeck Doryen , Rowlands David J. , Hogle James M. 
Published: December 2005

ABSTRACT
The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.
ICP0 Prevents RNase L-Independent rRNA Cleavage in Herpes Simplex Virus Type 1-Infected Cells
Authors: Sobol Paul T. , Mossman Karen L. 
Published: December 2005

ABSTRACT
The classical interferon (IFN)-dependent antiviral response to viral infection involves the regulation of IFN-stimulated genes (ISGs), one being the gene encoding cellular endoribonuclease RNase L, which arrests protein synthesis and induces apoptosis by nonspecifically cleaving rRNA. Recently, the herpes simplex virus type 1 (HSV-1) protein ICP0 has been shown to block the induction of ISGs by subverting the IFN pathway upstream of the 2′-5′-oligoadenylate synthetase (OAS)/RNase L pathway. We report that ICP0 also prevents rRNA degradation at late stages of HSV-1 infection, independent of its E3 ubiquitin ligase activity, and that the resultant rRNA degradation is independent of the classical RNase L antiviral pathway. Moreover, the degradation is independent of the viral RNase vhs and is independent of IFN response factor 3. These studies indicate the existence of another, previously unidentified, RNase that is part of the host antiviral response to viral infection.
Identification of a Novel Pathway Essential for the Immediate-Early, Interferon-Independent Antiviral Response to Enveloped Virions
Authors: Noyce Ryan S. , Collins Susan E. , Mossman Karen L. 
Published: December 2005

ABSTRACT
Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immunity. Previously we showed that entry of virion particles from a diverse array of enveloped virus families was capable of eliciting an interferon regulatory factor 3 (IRF-3)-mediated antiviral state in human fibroblasts in the absence of interferon production. Here we show that extracellular regulated kinase 1/2, p38 mitogen-activated protein kinase, and Jun N-terminal kinase/stress-activated protein kinase activities are not required for antiviral state induction. In contrast, treatment of cells with LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3 kinase) family, prevents the induction of interferon-stimulated gene 56 (ISG56) and an antiviral response upon entry of virus particles. However, the prototypic class I p85/p110 PI3 kinase and its downstream effector Akt/PKB are dispensable for ISG and antiviral state induction. Furthermore, DNA-PK and PAK1, LY294002-sensitive members of the PI3 kinase family shown previously to be involved in IRF-3 activation, are also dispensable for ISG and antiviral state induction. The LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear translocation upon virus particle entry. Together, these data suggest that virus entry triggers an innate antiviral response that requires the activity of a novel PI3 kinase family member.
Defective Assembly of Influenza A Virus due to a Mutation in the Polymerase Subunit PA
Authors: Regan John F. , Liang Yuying , Parslow Tristram G. 
Published: December 2005

ABSTRACT
The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.
Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 and PIASy Mediates SUMO-1 Addition Required Early in Infection
Authors: Yueh Andrew , Leung Juliana , Bhattacharyya Subarna , Perrone Lucy A. , de los Santos Kenia , Pu Szy-yuan , Goff Stephen P. 
Published: December 2005

ABSTRACT
Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier (SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leukemia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to a consensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped to residues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1 protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines near the Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation. Introduction of these mutations into the complete viral genome blocked virus replication. The mutants exhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutant viruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but were unable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The results suggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for early events of infection, after reverse transcription and before nuclear entry and viral DNA integration.
Herpes Simplex Virus DNA Synthesis Is Not a Decisive Regulatory Event in the Initiation of Lytic Viral Protein Expression in Neurons In Vivo during Primary Infection or Reactivation from Latency
Authors: Sawtell N. M. , Thompson R. L. , Haas R. L. 
Published: December 2005

ABSTRACT
The herpes simplex virus genome can enter a repressed transcriptional state (latency) in sensory neurons of the host nervous system. Although reduced permissiveness of the neuronal environment is widely accepted as a causal factor, the molecular pathway(s) directing and maintaining the viral genome in the latent state remains undefined. Over the past decade, the field has been strongly influenced by the observations of Kosz-Vnenchak et al., which have been interpreted to indicate that, in sensory neurons in vivo, a critical level of viral DNA synthesis within the neuron is required for sufficient viral immediate-early (IE) and early (E) gene expression (M. Kosz-Vnenchak, J. Jacobson, D. M. Coen, and D. M. Knipe, J. Virol. 67:5383-5393, 1993). The levels of IE and E genes are, in turn, thought to regulate the decision to enter the lytic cycle or latency. We have reexamined this issue using new strategies for in situ detection and quantification of viral gene expression in whole tissues. Our results using thymidine kinase-null and rescued mutants as well as wild-type strains in conjunction with viral DNA synthesis blockers demonstrate that (i) despite inhibition of viral DNA replication, many neurons express lytic viral proteins, including IE proteins, during acute infection in the ganglion; (ii) at early times postinoculation, the number of neurons expressing viral proteins in the ganglion is not reduced by inhibition of viral DNA replication; and (iii) following a reactivation stimulus, the numbers of neurons and apparent levels of lytic viral proteins, including IE proteins, are not reduced by inhibition of viral DNA replication. We conclude that viral DNA replication in the neuron per se does not regulate IE gene expression or entry into the lytic cycle.