bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunization of Mice with a TolA-Like Surface Protein ofTrypanosoma cruziGenerates CD4+T-Cell-Dependent Parasiticidal Activity
Authors: Quanquin Natalie M. , Galaviz Charles , Fouts David L. , Wrightsman Ruth A. , Manning Jerry E. 
Published: December 2019

ABSTRACTThe gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form ofTrypanosoma cruzihas been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins ofEscherichia coliandPseudomonas aeruginosa. Based on these similarities, we have designated this gene familytolT. Immunization of mice with recombinant TolT generates a population of CD4+T lymphocytes that recognizeT. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-γ), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN-γ and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected withT. cruzialso produce significant levels of IFN-γ when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected withT. cruzi.
Lactoferrin-Lipid A-Lipopolysaccharide Interaction: Inhibition by Anti-Human Lactoferrin Monoclonal Antibody AGM 10.14
Authors: Caccavo Domenico , Afeltra Antonella , Pece Salvatore , Giuliani Giuseppe , Freudenberg Marina , Galanos Chris , Jirillo Emilio 
Published: December 2019

ABSTRACTLactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.
Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
Authors: Condon Michael R. , Viera Alejandro , D’Alessio Michael , Diamond Gill 
Published: December 2019

ABSTRACTMulticellular organisms utilize a battery of extracellular and cellular mechanisms to defend against microbial infiltration. Among the armamentarium used by the small intestine to defend against microbial invasion are antimicrobial peptides called defensins. We previously have shown that gut barrier function is impaired following hemorrhagic shock, resulting in translocation of bacteria or endotoxin. Using a rat model, we examined the effect of hemorrhagic shock on α-defensin expression. We utilized the anchored reverse transcriptase PCR strategy to isolate a rat enteric defensin cDNA. The cDNA is 406 bases in length and encodes a putative prepro-enteric defensin that we have named rat defensin 5 (RD-5). RD-5 expression is restricted to the small intestine and is specifically localized by in situ hybridization to the Paneth cells. A 10-fold increase in its steady state levels was observed in the distal intestine immediately after the termination of shock. This is the first study to show that enteric defensins are inducible following injury. We suggest that enteric defensins may contribute to the complex and integrated barrier function of the intestinal mucosal surface.
Cell Surface-Exposed Tetanus Toxin Fragment C Produced by RecombinantBacillus anthracisProtects against Tetanus Toxin
Authors: Mesnage Stéphane , Weber-Levy Martine , Haustant Michel , Mock Michèle , Fouet Agnès 
Published: December 2019

ABSTRACTBacillus anthracis, the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinantB. anthracisstrain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.
The 102-KilobasepgmLocus ofYersinia pestis: Sequence Analysis and Comparison of Selected Regions among DifferentYersinia pestisandYersinia pseudotuberculosisStrains
Authors: Buchrieser Carmen , Rusniok Christophe , Frangeul Lionel , Couve Elisabeth , Billault Alain , Kunst Frank , Carniel Elisabeth , Glaser Philippe 
Published: December 2019

ABSTRACTWe report the complete 119,443-bp sequence of thepgmlocus fromYersinia pestisand its flanking regions. Sequence analysis confirms that the 102-kb unstablepgmlocus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system ofBordetella pertussis. Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among differentY. pestisandYersinia pseudotuberculosisstrains, the sequence of selected regions of thepgmlocus and flanking regions were compared from 20 differentY. pestisand 10Y. pseudotuberculosisstrains. The results showed that the genes interrupted inY. pestisare intact inY. pseudotuberculosis. However, one of these mutations, in thebvgShomologue, is only present inY. pestisstrains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29Yersiniastrains suggest thatY. pestisis a recently emerged pathogen that is probably entering the initial phase of reductive evolution.
Control of Filament Formation in
Candida albicans
by Polyamine Levels
Authors: Herrero Ana B. , López M. Carmen , García Susana , Schmidt Axel , Spaltmann Frank , Ruiz-Herrera José , Dominguez Angel 
Published: December 2019

ABSTRACT

Candida albicans
, the most common fungal pathogen, regulates its cellular morphology in response to environmental conditions. The
ODC
gene, which encodes ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was isolated and disrupted. Homozygous null
Candida
mutants behaved as polyamine auxotrophs and grew exclusively in the yeast form at low polyamine levels (0.01 mM putrescine) under all conditions tested. An increase in the polyamine concentration (10 mM putrescine) restored the capacity to switch from the yeast to the filamentous form. The strain with a deletion mutation also showed increased sensitivity to salts and calcofluor white. This
Candida odc/odc
mutant was virulent in a mouse model. The results suggest a model in which polyamine levels exert a pleiotrophic effect on transcriptional activity.
Dynamics of Iron Uptake and Fe3O4Biomineralization during Aerobic and Microaerobic Growth ofMagnetospirillum gryphiswaldense
Authors: Schüler Dirk , Baeuerlein Edmund 
Published: December 2019

ABSTRACTIron uptake and magnetite (Fe3O4) crystal formation could be studied in the microaerophilic magnetic bacteriumMagnetospirillum gryphiswaldenseby using a radioactive tracer method for iron transport and a differential light-scattering technique for magnetism. Magnetite formation occurred only in a narrow range of low oxygen concentration, i.e., 2 to 7 μM O2at 30°C. Magnetic cells stored up to 2% iron as magnetite crystals in intracytoplasmic vesicles. This extraordinary uptake of iron was coupled tightly to the biomineralization of up to 60 magnetite crystals with diameters of 42 to 45 nm.
Identification and Characterization of a New Organic Hydroperoxide Resistance (
ohr
) Gene with a Novel Pattern of Oxidative Stress Regulation from
Xanthomonas campestris
pv. phaseoli
Authors: Mongkolsuk Skorn , Praituan Wipa , Loprasert Suvit , Fuangthong Mayuree , Chamnongpol Sangpen 
Published: December 2019

ABSTRACT

We have isolated a new organic hydroperoxide resistance (
ohr
) gene from
Xanthomonas campestris
pv. phaseoli. This was done by complementation of an
Escherichia coli
alkyl hydroperoxide reductase mutant with an organic hydroperoxide-hypersensitive phenotype.
ohr
encodes a 14.5-kDa protein. Its amino acid sequence shows high homology with several proteins of unknown function. An
ohr
mutant was subsequently constructed, and it showed increased sensitivity to both growth-inhibitory and killing concentrations of organic hydroperoxides but not to either H
2
O
2
or superoxide generators. No alterations in sensitivity to other oxidants or stresses were observed in the mutant.
ohr
had interesting expression patterns in response to low concentrations of oxidants. It was highly induced by organic hydroperoxides, weakly induced by H
2
O
2
, and not induced at all by a superoxide generator. The novel regulation pattern of
ohr
suggests the existence of a second organic hydroperoxide-inducible system that differs from the global peroxide regulator system, OxyR. Expression of
ohr
in various bacteria tested conferred increased resistance to
tert
-butyl hydroperoxide killing, but this was not so for wild-type
Xanthomonas
strains. The organic hydroperoxide hypersensitivity of
ohr
mutants could be fully complemented by expression of
ohr
or a combination of
ahpC
and
ahpF
and could be partially complemented by expression
ahpC
alone. The data suggested that Ohr was a new type of organic hydroperoxide detoxification protein.
Isolation and Characterization of
Methanobacterium thermoautotrophicum
ΔH Mutants Unable To Grow under Hydrogen-Deprived Conditions
Authors: Pennings Jeroen L. A. , Keltjens Jan T. , Vogels Godfried D. 
Published: December 2019

ABSTRACT

By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of
Methanobacterium thermoautotrophicum
that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F
420
-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F
390
, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.
Acquired Thermotolerance and Temperature-Induced Protein Accumulation in the Extremely Thermophilic Bacterium
Rhodothermus obamensis
Authors: Takai Ken , Nunoura Takuro , Sako Yoshihiko , Uchida Aritsune 
Published: December 2019

ABSTRACT

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium
Rhodothermus obamensis
. The survival at temperatures superoptimal for growth (90 and 95°C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.
Paradoxical Enhancement of the Activity of a Bacterial Multidrug Transporter Caused by Substitutions of a Conserved Residue
Authors: Klyachko Katya A. , Neyfakh Alexander A. 
Published: December 2019

ABSTRACT

Substitution of threonine or serine for the evolutionary conserved intramembrane proline P
347
of the
Bacillus subtilis
multidrug transporter Bmr significantly increases the toxin-effluxing activity of Bmr without affecting its abundance in the cell. In cocultivation experiments, we demonstrate that although the mutant T
347
Bmr is advantageous to cells growing in the presence of a toxin, the wild-type P
347
Bmr is advantageous under the conditions of nutritional limitation. This may explain why Bmr has evolved the way it did, that is, with proline at position 347. These observations provide a basis for speculating that the evolution of Bmr has been determined by its presently unidentified natural function rather than by its ability to expel diverse toxins from the cell.
Molecular Characterization of the Gene Encoding the DNA Gyrase A Subunit of
Streptococcus pneumoniae
Authors: Balas Delia , Fernández-Moreira Esteban , De La Campa Adela G. 
Published: December 2019

ABSTRACT

The gene encoding the DNA gyrase A subunit of
Streptococcus pneumoniae
was cloned and sequenced. The
gyrA
gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an
Escherichia coli
expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of
gyrA
. The bend center was mapped and located between the −35 and −10 regions of the promoter. Primer extension analysis showed that
gyrA
transcription initiates 6 bp downstream of an extended −10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of
gyrA
are discussed.
Characterization of the
hca
Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in
Escherichia coli
K-12
Authors: Díaz Eduardo , Ferrández Abel , García José L. 
Published: December 2019

ABSTRACT

We have identified, cloned, and sequenced the
hca
cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in
Escherichia coli
K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (
hcaA1A2CBD
) and two additional genes transcribed in the opposite direction that encode a potential permease (
hcaT
) and a regulator (
hcaR
). Sequence comparisons revealed that while
hcaA1A2CD
genes encode the four subunits of the 3-phenylpropionate dioxygenase, the
hcaB
gene codes for the corresponding
cis
-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in
E. coli
. The inducible expression of the
hca
genes requires the presence of the
hcaR
gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2,3-dihydroxy derivative. Further catabolism of DHPP requires the
mhp
-encoded
meta
fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. Garcı́a, and E. Dı́az, J. Bacteriol. 179:2573–2581, 1997). Expression in
Salmonella typhimurium
of the
mhp
genes alone or in combination with the
hca
cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent
mhp
- and
hca
-encoded pathways are also functional in
S. typhimurium
, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.
In Vitro Synthesis of Multicopy Single-Stranded DNA, Using Separate Primer and Template RNAs, by
Escherichia coli
Reverse Transcriptase
Authors: Shimamoto Tadashi , Kawanishi Hideki , Tsuchiya Tomofusa , Inouye Sumiko , Inouye Masayori 
Published: December 2019

ABSTRACT

A minor population of wild strains of
Escherichia coli
contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA). The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the
msr-msd
region under a single promoter. A single RNA transcript from the
msr-msd
region serves not only as a template but also as a primer for msDNA synthesis. Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed
msr
and
msd
transcripts. DNA sequencing of the cell-free product revealed that the sequence of the 5′-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2′-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73. The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the
msr
RNA transcript.
An Isoflavonoid-Inducible Efflux Pump in
Agrobacterium tumefaciens
Is Involved in Competitive Colonization of Roots
Authors: Palumbo Jeffrey D. , Kado Clarence I. , Phillips Donald A. 
Published: December 2019

ABSTRACT

Agrobacterium tumefaciens
1D1609, which was originally isolated from alfalfa (
Medicago sativa
L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn
5
-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame,
ifeA
, of a three-gene locus (
ifeA
,
ifeB
, and
ifeR
), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (
P
≤ 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (
ifeA
::
gusA
) and I-6 (
ifeA
::Ω-Tc), confirmed the importance of
ifeA
for competitive root colonization. Exposure to the isoflavonoid coumestrol increased β-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the
ifeA-gusA
reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on
A. tumefaciens
in an environment where the inducing molecules are known to be present.
Effects of Carbon Source on Expression of F
o
Genes and on the Stoichiometry of the c Subunit in the F
1
F
o
ATPase of
Escherichia coli
Authors: Schemidt Randy A. , Qu Jun , Williams James R. , Brusilow William S. A. 
Published: December 2019

ABSTRACT

Expression of the genes for the membrane-bound F
o
sector of the
Escherichia coli
F
1
F
o
proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric F
o
proton channel. Transcriptional and translational
lacZ
fusions to the promoter and to two F
o
genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of
uncB
, encoding the a subunit of F
o
, are higher than during growth on glucose. In contrast, translation of the
uncE
gene, encoding the c subunit of F
o
, is higher during growth on glucose than during growth on succinate. Translation rates of both
uncB
and
uncE
change as culture density increases, but transcription rates do not. Quantitation of the c stoichiometry shows that more c subunits are assembled into the F
1
F
o
ATPase in cells grown on glucose than in cells grown on succinate.
E. coli
therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.
The
MTCY428.08
Gene of
Mycobacterium tuberculosis
Codes for NAD
+
Synthetase
Authors: Cantoni Rita , Branzoni Manuela , Labò Monica , Rizzi Menico , Riccardi Giovanna 
Published: December 2019

ABSTRACT

The product of the
MTCY428.08
gene of
Mycobacterium tuberculosis
shows sequence homology with several NAD
+
synthetases. The
MTCY428.08
gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in
Escherichia coli
led to overproduction of glutathione
S
-transferase fused and His
6
-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the
MTCY428.08
gene of
M. tuberculosis
is the structural gene for NAD
+
synthetase.