bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pal Lipoprotein ofEscherichia coliPlays a Major Role in Outer Membrane Integrity
Authors: Cascales Eric , Bernadac Alain , Gavioli Marthe , Lazzaroni Jean-Claude , Lloubes Roland 
Published: April 2009

ABSTRACTThe Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared inEscherichia coli. lppandtol-palmutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of anlppstrain but that overproduced Lpp has no effect in apalstrain. Together with the previously reported observation that overproduced TolA complements anlppbut not apalstrain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.
Role of GlnK in NifL-Mediated Regulation of NifA Activity inAzotobacter vinelandii
Authors: Rudnick Paul , Kunz Christopher , Gunatilaka Malkanthi K. , Hines Eric R. , Kennedy Christina 
Published: April 2009

ABSTRACTIn several diazotrophic species ofProteobacteria, PIIsignal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH4+by several mechanisms. InAzotobacter vinelandii, expression ofnifA, encoding thenif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the PII-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP inA. vinelandiiwere studied to determine whether the Nif−phenotype ofglnDstrains was due to an inability to modify GlnK, an effort previously hampered becauseglnKis an essential gene in this organism. AglnKY51Fmutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH4+, suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation.glnKY51Fstrains were significantly impaired for diazotrophic growth and expression of anifH-lacZfusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041–6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacteriumKlebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.
Shigella
Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length
Authors: Tamano Koichi , Katayama Eisaku , Toyotome Takahito , Sasakawa Chihiro 
Published: April 2009

ABSTRACT

The
Shigella
type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In
S. flexneri
, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the
spa
genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the
spa32
gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned
spa32
into the
spa32
mutant, the bacteria produced needles of wild-type length. The
spa32
mutant overexpressing MxiH produced extremely long (>5 μm) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The
spa32
mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of
Salmonella invJ
, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the
spa32
mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in
Shigella
spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of
Salmonella
InvJ.
λ Integrase Complementation at the Level of DNA Binding and Complex Formation
Authors: Nunes-Düby Simone E. , Radman-Livaja Marta , Kuimelis Robert G. , Pearline Rachel V. , McLaughlin Larry W. , Landy Arthur 
Published: April 2009

ABSTRACTSite-specific recombinases of the λ Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer incis(as for Cre recombinase), or it may recruit the tyrosine from its partner intransto form a composite active site (as for Flp recombinase). The crystal structure of the λ Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on λ integrase (Int) generated data that were not in agreement. Support forcisandtranscleavage came from assays with bispecific DNA substrates for λ and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence forciscleavage, but they strongly suggest that the previously described complementation results cannot be used in support of atrans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-orderattL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (incis) by mutant λ Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the λ Int mutants.
Disruption oflolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal forEscherichia coliand Prevents Release of Lipoproteins from the Inner Membrane
Authors: Narita Shin-ichiro , Tanaka Kimie , Matsuyama Shin-ichi , Tokuda Hajime 
Published: April 2009

ABSTRACTATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane ofEscherichia coli. Mutations resulting in defective LolD were previously shown to be lethal forE. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release. To address these issues, a chromosomallolC-lolD-lolEnull mutant harboring a helper plasmid that carries thelolCDEgenes and a temperature-sensitive replicon was constructed. The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE. In addition to functional LolD, both LolC and LolE were required for growth. At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane. Furthermore, both LolC and LolE were essential for the release of lipoproteins. On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein. From these results, we conclude that the LolCDE complex is an essential ABC transporter forE. coliand the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane.
Mutations in the Thiol-Disulfide Oxidoreductases BdbC and BdbD Can Suppress CytochromecDeficiency of CcdA-DefectiveBacillus subtilisCells
Authors: Erlendsson Lýđur S. , Hederstedt Lars 
Published: April 2009

ABSTRACTCytochromes of thectype in the gram-positive bacteriumBacillus subtilisare all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochromecmust be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochromecand spore synthesis. We have discovered that mutations in thebdbCandbdbDgenes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that theseB. subtilisproteins functionally correspond to the well-characterizedEscherichia coliDsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.
The Flavoenzyme Ferredoxin (Flavodoxin)-NADP(H) Reductase Modulates NADP(H) Homeostasis during the
soxRS
Response of
Escherichia coli
Authors: Krapp Adriana R. , Rodriguez Ramiro E. , Poli Hugo O. , Paladini Darío H. , Palatnik Javier F. , Carrillo Néstor 
Published: April 2009

ABSTRACT

Escherichia coli
cells from strain
fpr
, deficient in the
soxRS
-induced ferredoxin (flavodoxin)-NADP(H) reductase (FPR), display abnormal sensitivity to the bactericidal effects of the superoxide-generating reagent methyl viologen (MV). Neither bacteriostatic effects nor inactivation of oxidant-sensitive hydrolyases could be detected in
fpr
cells exposed to MV. FPR inactivation did not affect the MV-driven
soxRS
response, whereas FPR overexpression led to enhanced stimulation of the regulon, with concomitant oxidation of the NADPH pool. Accumulation of a site-directed FPR mutant that uses NAD(H) instead of NADP(H) had no effect on
soxRS
induction and failed to protect
fpr
cells from MV toxicity, suggesting that FPR contributes to NADP(H) homeostasis in stressed bacteria.
Sensitive Screening Tests for Suspected Class A Carbapenemase Production in Species of
Enterobacteriaceae
Authors: Pasteran Fernando , Mendez Tania , Guerriero Leonor , Rapoport Melina , Corso Alejandra 
Published: April 2009

ABSTRACT

The detection of class A serine-carbapenemases among species of
Enterobacteriaceae
remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated
Enterobacteriaceae
strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of
Enterobacteriaceae
was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of β-lactamases (extended-spectrum β-lactamases, AmpCs, and metallo-β-lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control.
Enrichment Broth Improved Detection of Extended-Spectrum-Beta-Lactamase-Producing Bacteria in Throat and Rectal Surveillance Cultures of Samples from Patients in Intensive Care Units
Authors: Murk Jean-Luc A. N. , Heddema Edou R. , Hess Dorine L. J. , Bogaards Johannes A. , Vandenbroucke-Grauls Christina M. J. E. , Debets-Ossenkopp Yvette J. 
Published: April 2009

ABSTRACT

We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-beta-lactamase-producing (ESBL
+
) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL
+
bacteria detected in samples obtained from patients in our intensive care unit.
Genetic Variability of Group A Human Respiratory Syncytial Virus Strains Circulating in Germany from 1998 to 2007
Authors: Reiche Janine , Schweiger Brunhilde 
Published: April 2009

ABSTRACT
The variability between respiratory syncytial virus (RSV) strains is one of the features of RSV infections that might contribute to the ability of the virus to infect people repeatedly and cause yearly outbreaks. To study the molecular epidemiology of RSV, more than 1,400 RSV isolates from human nasopharyngeal aspirates or nasal or throat swabs from patients with respiratory illness were identified and differentiated by TaqMan reverse transcription-PCR into groups A and B. RSV group A was dominant in seven out of nine epidemic seasons. Phylogenetic analysis revealed that RSV group A genotypes GA2 and GA5 circulated from 1998 to 2007. Genotype GA7 was present in only two seasons (1999 to 2000 and 2002 to 2003). Comparison of the synonymous mutation/nonsynonymous mutation ratios showed greater evidence for selection pressure for genotype GA2 (1.18) than for GA5 (4.34). Partial protein sequences were predicted to encode G proteins of 298 amino acids in length and in a few cases of G proteins of 297 amino acids in length. Amino acid analysis also revealed genotype-specific amino acid substitutions: two substitutions for genotype GA2, seven for GA5, and three for GA7. Two to four putative, genotype-specific N-linked glycosylation sites were determined. Predicted O-glycosylation sites included 22 to 34 residues. This study provides for the first time data on the circulation pattern of RSV group A genotypes and their molecular characterization in Germany during nine consecutive epidemic seasons.
HLA Class I Subtype-Dependent Expansion of KIR3DS1
+
and KIR3DL1
+
NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection
Authors: Alter Galit , Rihn Suzannah , Walter Katharine , Nolting Anne , Martin Maureen , Rosenberg Eric S. , Miller Jeffrey S. , Carrington Mary , Altfeld Marcus 
Published: April 2009

ABSTRACT

NK cells are critical in the early containment of viral infections. Epidemiological and functional studies have shown an important role of NK cells expressing specific killer immunoglobulin-like receptors (KIRs) in the control of human immunodeficiency virus type 1 (HIV-1) infection, but little is known about the mechanisms that determine the expansion of these antiviral NK cell populations during acute HIV-1 infection. Here we demonstrate that NK cells expressing the activating receptor KIR3DS1
+
and, to a lesser extent, the inhibitory receptor KIR3DL1
+
specifically expand in acute HIV-1 infection in the presence of HLA-B Bw480I, the putative HLA class I ligand for KIR3DL1/3DS1. These data demonstrate for the first time the HLA class I subtype-dependent expansion of specific KIR
+
NK cells during an acute viral infection in humans.
Identification of Infected B-Cell Populations by Using a Recombinant Murine Gammaherpesvirus 68 Expressing a Fluorescent Protein
Authors: Collins Christopher M. , Boss Jeremy M. , Speck Samuel H. 
Published: April 2009

ABSTRACT
Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.
Infection of HLA-DR1 Transgenic Mice with a Human Isolate of Influenza A Virus (H1N1) Primes a Diverse CD4 T-Cell Repertoire That Includes CD4 T Cells with Heterosubtypic Cross-Reactivity to Avian (H5N1) Influenza Virus
Authors: Richards Katherine A. , Chaves Francisco A. , Sant Andrea J. 
Published: April 2009

ABSTRACTThe specificity of the CD4 T-cell immune response to influenza virus is influenced by the genetic complexity of the virus and periodic encounters with variant subtypes and strains. In order to understand what controls CD4 T-cell reactivity to influenza virus proteins and how the influenza virus-specific memory compartment is shaped over time, it is first necessary to understand the diversity of the primary CD4 T-cell response. In the study reported here, we have used an unbiased approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells, hemagglutinin (HA), neuraminidase (NA), nucleoprotein, and the NS1 protein, which is expressed in infected cells but excluded from virion particles. Our studies revealed an extensive diversity of influenza virus-specific CD4 T cells that includes T cells for each viral protein and for the unexpected immunogenicity of the NS1 protein. Due to the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody to influenza virus, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited by a circulating H1N1 strain to cross-react with related sequences found in an avian H5N1 virus and find substantial cross-reactivity, suggesting that seasonal vaccines may help promote protection against avian influenza virus.
A Point Mutation, E95D, in the Mumps Virus V Protein Disengages STAT3 Targeting from STAT1 Targeting
Authors: Puri Mamta , Lemon Ken , Duprex W. Paul , Rima Bertus K. , Horvath Curt M. 
Published: April 2009

ABSTRACT

Mumps virus, like other paramyxoviruses in the
Rubulavirus
genus, encodes a V protein that can assemble a ubiquitin ligase complex from cellular components, leading to the destruction of cellular signal transducer and activator of transcription (STAT) proteins. While many V proteins target the interferon-activated STAT1 or STAT2 protein, mumps virus V protein is unique in its ability to also target STAT3 for ubiquitin modification and proteasome-mediated degradation. Here we report that a single amino acid substitution in the mumps virus V protein, E95D, results in defective STAT3 targeting while maintaining the ability to target STAT1. Results indicate that the E95D mutation disrupts the ability of the V protein to associate with STAT3. A recombinant mumps virus carrying the E95D mutation in its P and V proteins replicates normally in cultured cells but fails to induce targeting of STAT3. Infection with the recombinant virus results in the differential regulation of a number of cellular genes compared to wild-type mumps virus and increases cell death in infected cells, producing a large-plaque phenotype.