bluuebunny/crossref_metadata_2025_split huggingface dataset:
The REP2 Repeats of the Genome ofNeisseria meningitidisAre Associated with Genes Coordinately Regulated during Bacterial Cell Interaction
Authors: Morelle Sandrine , Carbonnelle Etienne , Nassif Xavier 
Published: April 2003

ABSTRACTInteraction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence ofNeisseria meningitidisZ2491 revealed the presence of many repeats. One of these, designated REP2, contains a −24/−12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes arepilC1andcrgA, described as being involved in steps essential for the interaction ofN. meningitidiswith host cells. Furthermore, the REP2 sequences located upstream ofpilC1andcrgAcorrespond to the previously identified promoters known to be induced during the initial localized adhesion ofN. meningitidiswith human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances aspilC1andcrgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion ofN. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.
The Mechanism of Bacterial Infection by Filamentous Phages Involves Molecular Interactions between TolA and Phage Protein 3 Domains
Authors: Karlsson Fredrik , Borrebaeck Carl A. K. , Nilsson Nina , Malmborg-Hager Ann-Christin 
Published: April 2003

ABSTRACTThe early events in filamentous bacteriophage infection of gram-negative bacteria are mediated by the gene 3 protein (g3p) of the virus. This protein has a sophisticated domain organization consisting of two N-terminal domains and one C-terminal domain, separated by flexible linkers. The molecular interactions between these domains and the known bacterial coreceptor protein (TolA) were studied using a biosensor technique, and we report here on interactions of the viral coat protein with TolA, as well as on interactions between the TolA molecules. We detected an interaction between the pilus binding second domain (N2) of protein 3 and the bacterial TolA. This novel interaction was found to depend on the periplasmatic domain of TolA (TolAII). Furthermore, extensive interaction was detected between TolA molecules, demonstrating that bacterial TolA has the ability to interact functionally with itself during phage infection. The kinetics of g3p binding to TolA is also different from that of bacteriocins, since both N-terminal domains of g3p were found to interact with TolA. The multiple roles for each of the separate g3p and TolA domains imply a delicate interaction network during the phage infection process and a model for the infection mechanism is hypothesized.
The
Staphylococcus aureus cidAB
Operon: Evaluation of Its Role in Regulation of Murein Hydrolase Activity and Penicillin Tolerance
Authors: Rice Kelly C. , Firek Brian A. , Nelson Jeremy B. , Yang Soo-Jin , Patton Toni G. , Bayles Kenneth W. 
Published: April 2003

ABSTRACT

Recent studies have shown that expression of the
Staphylococcus aureus lrgAB
operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. It was proposed that the
lrgAB
gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell. In the present study the
cidAB
operon was identified and characterized based on the similarity of the
cidA
and
cidB
gene products to the products of the
lrgAB
operon. Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the
cidA
gene results in decreased extracellular murein hydrolase activity compared to that of
S
.
aureus
RN6390, the parental strain. Complementation of
cidA
expression restored the wild-type phenotype, indicating that expression of the
cidAB
operon has a positive influence on extracellular murein hydrolase activity. The
cidA
mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin. However, complementation of the
cidA
defect did not restore penicillin sensitivity to wild-type levels. Reverse transcriptase PCR also revealed that
cidAB
is maximally expressed during early exponential growth, opposite of what was previously observed for
lrgAB
expression. Based on these results, we propose that the
cidAB
operon encodes the holin-like counterpart of the
lrgAB
operon and acts in a manner opposite from that of
lrgAB
by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing.
Phenotypic Mutants of the Intracellular Actinomycete
Rhodococcus
equi
Created by In Vivo
Himar1
Transposon Mutagenesis
Authors: Ashour Joseph , Hondalus Mary K. 
Published: April 2003

ABSTRACT

Rhodococcus
equi
is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of
R
.
equi
disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a
Himar1
minitransposon delivered by a conditionally replicating plasmid for construction of
R
.
equi
mutants. We show that
Himar1
transposition in
R
.
equi
is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of
R
.
equi
. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type
R
.
equi
, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of
R
.
equi
virulence mechanisms and the creation of other attenuated strains with vaccine potential.
Mutations at Residues 282, 286, and 293 of Phage λ Integrase Exert Pathway-Specific Effects on Synapsis and Catalysis in Recombination
Authors: Bankhead Troy M. , Etzel Bernard J. , Wolven Felise , Bordenave Sylvain , Boldt Jeffrey L. , Larsen Teresa A. , Segall Anca M. 
Published: April 2003

ABSTRACTBacteriophage λ integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. Theattsites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: theattLsite probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA withattR.
Global Analysis of Genes Regulated by EvgA of the Two-Component Regulatory System in
Escherichia coli
Authors: Nishino Kunihiko , Inazumi Yoshihiko , Yamaguchi Akihito 
Published: April 2003

ABSTRACT

The response regulator EvgA controls expression of multiple genes conferring antibiotic resistance in
Escherichia coli
(K. Nishino and A. Yamaguchi, J. Bacteriol. 184:2319-2323, 2002). To understand the whole picture of EvgA regulation, DNA macroarray analysis of the effect of EvgA overproduction was performed. EvgA activated genes related to acid resistance, osmotic adaptation, and drug resistance.
In Vitro Properties of RpoS (σ
S
) Mutants of
Escherichia coli
with Postulated N-Terminal Subregion 1.1 or C-Terminal Region 4 Deleted
Authors: Gowrishankar J. , Yamamoto Kaneyoshi , Subbarayan P. R. , Ishihama Akira 
Published: April 2003

ABSTRACT

Derivatives of the stationary-phase sigma factor σ
S
of
Escherichia coli
lacking either of two conserved domains, the postulated N-terminal subregion 1.1 or the C-terminal region 4, were shown to be competent in vitro for transcription initiation from several σ
S
-dependent promoters on supercoiled DNA templates. Unlike wild-type σ
S
, however, the deletion derivatives were inactive on relaxed templates. The anomalous slow electrophoretic mobility of σ
S
on denaturing gels was corrected by deletion of subregion 1.1, suggesting that this domain in σ
S
may be structurally and functionally analogous to subregion 1.1 of σ
70
, substitutions in which have previously been shown to rectify the anomalous electrophoretic migration of σ
70
(V. Gopal and D. Chatterji, Eur. J. Biochem. 244:614-618, 1997).
Hydrogen-Oxidizing Capabilities ofHelicobacter hepaticusand In Vivo Availability of the Substrate
Authors: Maier Robert J. , Olson Jonathan , Olczak Adriana 
Published: April 2003

ABSTRACTHydrogen-oxidizing hydrogenase activity was detected inHelicobacter hepaticusand compared to the activity inHelicobacter pylorifor characteristics associated with hydrogen uptake respiratory hydrogenases. Intact whole cells could couple H2oxidation to oxygen uptake, and no H2uptake was observed without oxygen available to complete the respiratory pathway. TheH. hepaticusenzyme coupled H2oxidation to reduction of many positive potential acceptors, and it underwent anaerobic or reductive activation.H. hepaticushad a strong affinity for molecular H2(apparentKmof 2.5 μM), and microelectrode measurements on the livers of live mice demonstrated that H2is available in the host tissue at levels 20-fold greater than the apparent whole-cellKmvalue.
Heat Shock Protein 90-Independent Activation of Truncated Hepadnavirus Reverse Transcriptase
Authors: Wang Xingtai , Qian Xiaofeng , Guo Hwai-Chen , Hu Jianming 
Published: March 2003

ABSTRACT
The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed ε, and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and ε serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-ε interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its cochaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in ε interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive C-terminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-to-be-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors.
Silencing of the Baculovirus Op-
iap3
Gene by RNA Interference Reveals that It Is Required for Prevention of Apoptosis during
Orgyia pseudotsugata M
Nucleopolyhedrovirus Infection of Ld652Y Cells
Authors: Means John C. , Muro Israel , Clem Rollie J. 
Published: March 2003

ABSTRACT

The Op-
iap3
gene from the baculovirus
Orgyia pseudotsugata
M
nucleopolyhedrovirus (Op
M
NPV) inhibits apoptosis induced by a mutant of
Autographa californica
M
NPV (Ac
M
NPV) that lacks the antiapoptotic gene
p35
, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-
iap3
during Op
M
NPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during Op
M
NPV infection, we used RNA interference (RNAi) to silence Op-
iap3
expression during Op
M
NPV infection of Ld652Y cells. Infected cells treated with Op-
iap3
double-stranded RNA (dsRNA) did not accumulate detectable Op-
iap3
mRNA, confirming that the Op-
iap3
gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in Op
M
NPV-infected cells treated with Op-
iap3
dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-
iap3
dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during Op
M
NPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function.
Constitutive Inositol Phosphate Formation in Cytomegalovirus-Infected Human Fibroblasts Is due to Expression of the Chemokine Receptor Homologue pUS28
Authors: Minisini Rosalba , Tulone Calogero , Lüske Anke , Michel Detlef , Mertens Thomas , Gierschik Peter , Moepps Barbara 
Published: March 2003

ABSTRACT

An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1α, and the CX
3
C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca
2+
. Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via G
q/11
-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5′-O-(3-[
35
S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.
CXCL10 Production from Cytomegalovirus-Stimulated Microglia Is Regulated by both Human and Viral Interleukin-10
Authors: Cheeran Maxim C.-J. , Hu Shuxian , Sheng Wen S. , Peterson Phillip K. , Lokensgard James R. 
Published: March 2003

ABSTRACTGlial cells orchestrate immunocyte recruitment to focal areas of viral infection within the brain and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since recruitment of T lymphocytes plays a critical role in resolving cytomegalovirus (CMV) infection, we investigated the production of a T-cell chemoattractant, CXCL10 (gamma interferon-inducible protein 10) in response to viral infection of human glial cells. Infection with CMV was found to elicit the production of CXCL10 from primary microglial cells but not from astrocytes. This CXCL10 expression was not dependent on secondary protein synthesis but did require the phosphorylation of p38 mitogen-activated protein (MAP) kinase. In addition, migration of activated lymphocytes toward supernatants from CMV-stimulated microglial cells was partially suppressed by anti-CXCL10 antibodies. Since regulation of central nervous system inflammation is essential to allow viral clearance without immunopathology, microglial cells were then treated with anti-inflammatory cytokines. CMV-induced CXCL10 production from microglial cells was suppressed following treatment with interleukin-10 (IL-10) and IL-4 but not following treatment with transforming growth factor β. The IL-10-mediated inhibition of CXCL10 production was associated with decreased CMV-induced NF-κB activation but not decreased p38 MAP kinase phosphorylation. Finally, CMV infection of fully permissive astrocytes resulted in mRNA expression for the viral homologue to human IL-10 (i.e., cmvIL-10 [UL111a]) in its spliced form and conditioned medium from CMV-infected astrocytes inhibited virus-induced CXCL10 production from microglial cells through the IL-10 receptor. These findings present yet another mechanism through which CMV may subvert host immune responses.
Subcellular Localization of Feline Immunodeficiency Virus Integrase and Mapping of Its Karyophilic Determinant
Authors: Woodward Cora L. , Wang Yao , Dixon Wendy J. , Htun Han , Chow Samson A. 
Published: March 2003

ABSTRACT

Feline immunodeficiency virus (FIV), like other members of the lentivirus subfamily, such as human immunodeficiency virus type 1 (HIV-1), can infect nondividing and terminally differentiated cells. The transport of the preintegration complex into the nucleus is cell cycle-independent, but the mechanism is not well understood. Integrase is a key component of the complex and has been suggested to play a role in nuclear import during HIV-1 replication. To determine its karyophilic property, FIV integrase fused with glutathione
S
-transferase and enhanced green fluorescent protein was expressed in various feline and human cells and the subcellular localization was visualized by fluorescence microscopy. Wild-type FIV integrase was karyophilic in all cell lines tested and capable of targeting the fusion protein to the nuclei of transfected cells. Analysis of deletion and point mutation variants of FIV integrase failed to reveal any canonical nuclear localization signal, and the karyophilic determinant was mapped to the highly conserved N-terminal zinc-binding HHCC motif. A region near the C-terminal domain enriched with basic amino acid residues also affected the nuclear import of integrase. However, the role of this region is only modulatory in comparison to that of the zinc-binding domain. The N-terminal zinc-binding domain does not bind DNA and instead is essential in integrase multimerization. We therefore postulate that the karyophilic property of FIV integrase requires subunit multimerization promoted by the HHCC motif. Alternatively, the HHCC motif may directly promote interaction between FIV integrase and cellular proteins involved in nuclear import.
Switching Species Tropism: an Effective Way To Manipulate the Feline Coronavirus Genome
Authors: Haijema Bert Jan , Volders Haukeliene , Rottier Peter J. M. 
Published: March 2003

ABSTRACT
Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVΔ7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVΔ7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.