bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interactions between Coexisting Intracellular Genomes: Mitochondrial Density and
Wolbachia
Infection
Authors: Mouton L. , Henri H. , Fleury F. 
Published: January 2009

ABSTRACT

Many arthropods are infected with maternally transmitted microorganisms, leading to the coexistence of several intracellular genomes within the host cells, including their own mitochondria. As these genomes are cotransmitted, their patterns of evolution have been intimately linked, with possible consequences for the diversity and evolution of the host mitochondrial DNA. The evolutionary aspects of the situation have been thoroughly investigated, especially the selective sweep on the mitochondria as a result of
Wolbachia
invasion, whereas direct interactions between mitochondria and intracellular symbionts within the host cells or body have received little attention. Since endosymbionts exploit host resources but mitochondria supply energy to meet the bioenergetic demands of organisms, an unanswered question concerns the correlation between their densities. Here, we investigated the influence of
Wolbachia
symbiosis on mitochondrial density in two parasitic wasps of
Drosophila
species, both of which are naturally infected by three
Wolbachia
strains, but they differ in their degree of dependency on these bacteria. In
Leptopilina heterotoma
, all
Wolbachia
strains are facultative, whereas
Asobara tabida
requires a strain of
Wolbachia
for oogenesis to occur. In both species,
Wolbachia
infections are stable and well regulated, since the density of each strain does not depend on the presence or absence of other strains. Using lines that harbor various
Wolbachia
infection statuses, we found that mitochondrial density was not affected by the infection regardless of the sex and age of the host, which is strongly reminiscent of the independent regulation of specific
Wolbachia
strains and suggest that the protagonists coexist independently of each other as the result of a long-term coevolutionary interaction.
HacA-Dependent Transcriptional Switch Releases
hacA
mRNA from a Translational Block upon Endoplasmic Reticulum Stress
Authors: Mulder Harm J. , Nikolaev Igor 
Published: January 2009

ABSTRACT

Activation of the unfolded protein response (UPR) in eukaryotes involves the splicing of an unconventional intron from the mRNA encoding the transcriptional activator of the pathway. In
Saccharomyces cerevisiae
a 252-nucleotide (nt) unconventional intron is spliced out of the transcript of
HAC1
, changing the 3′ end of the
HAC1
open reading frame and relieving the transcript from a translational block in a single step. The translational block is caused by the base pairing of part of the unconventional intron with the 5′-untranslated region (5′UTR). In
Aspergillus niger
and other aspergilli, the unconventional intron in
hacA
mRNA is only 20 nt long. Since this intron is part of a stable stem-loop structure, base pairing with the 5′UTR, in contrast to the case with yeast
HAC1
, is not possible. However, analysis of the
hacA
mRNA revealed a GC-rich inverted repeat (18 base pairings). Upon the activation of the UPR, the 5′UTR of
hacA
mRNA is truncated by 230 nt, removing the left part of this inverted repeat. This implies a similar release of a translational block as in the case of
S. cerevisiae HAC1
but in two steps. The mechanism behind the 5′ truncation, which does not take place in either yeast
HAC1
or mammalian
xbp1
mRNA, has been hitherto unknown. Here we show that during secretion stress in
A. niger
,
hacA
transcription starts from a new start site closer to the ATG, relieving the transcript from translational attenuation. This transcriptional switch is mediated by HacA itself and the unfolded protein response element 2 (UPRE2) in the
hacA
promoter.
Analysis of All Protein Phosphatase Genes in
Aspergillus nidulans
Identifies a New Mitotic Regulator, Fcp1
Authors: Son Sunghun , Osmani Stephen A. 
Published: January 2009

ABSTRACT

Reversible protein phosphorylation is an important regulatory mechanism of cell cycle control in which protein phosphatases counteract the activities of protein kinases. In
Aspergillus nidulans
, 28 protein phosphatase catalytic subunit genes were identified. Systematic deletion analysis identified four essential phosphatases and four required for normal growth. Conditional alleles of these were generated using the
alcA
promoter. The deleted phosphatase strain collection and regulatable versions of the essential and near-essential phosphatases provide an important resource for further analysis of the role of reversible protein phosphorylation to the biology of
A. nidulans
. We further demonstrate that
nimT
and
bimG
have essential functions required for mitotic progression since their deletions led to classical G
2
- and M-phase arrest. Although not as obvious, cells with An
pphA
and An
nem1
deleted also have mitotic abnormalities. One of the essential phosphatases, the RNA polymerase II C-terminal domain phosphatase An
fcp1
, was further examined for potential functions in mitosis because a temperature-sensitive An
fcp1
allele was isolated in a genetic screen showing synthetic interaction with the
cdk1F
mutation, a hyperactive mitotic kinase. The An
fcp1

ts

cdk1F
double mutant had severe mitotic defects, including inability of nuclei to complete mitosis in a normal fashion. The severity of the An
fcp1

ts

cdk1F
mitotic phenotypes were far greater than either single mutant, confirming the synthetic nature of their genetic interaction. The mitotic defects of the An
fcp1

ts

cdk1F
double mutant suggests a previously unrealized function for AnFCP1 in regulating mitotic progression, perhaps counteracting Cdk1-mediated phosphorylation.
Isolation and Characterization of
Cryptococcus neoformans
Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis
Authors: Botts Michael R. , Giles Steven S. , Gates Marcellene A. , Kozel Thomas R. , Hull Christina M. 
Published: January 2009

ABSTRACT

Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus
Cryptococcus neoformans
. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for
C. neoformans
virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that
C. neoformans
spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.
Identification and Specific Localization of Tyrosine-Phosphorylated Proteins in
Trypanosoma brucei
Authors: Nett Isabelle R. E. , Davidson Lindsay , Lamont Douglas , Ferguson Michael A. J. 
Published: January 2009

ABSTRACT

Phosphorylation on tyrosine residues is a key signal transduction mechanism known to regulate intercellular and intracellular communication in multicellular organisms. Despite the lack of conventional tyrosine kinases in the genome of the single cell organism
Trypanosoma brucei
, phosphorylation on trypanosomal protein tyrosine residues has been reported for this parasite. However, the identities of most of the tyrosine-phosphorylated proteins and their precise site(s) of phosphorylation were unknown. Here, we have applied a phosphotyrosine-specific proteomics approach to identify 34 phosphotyrosine-containing proteins from whole-cell extracts of procyclic form
T. brucei
. A significant proportion of the phosphotyrosine-containing proteins identified in this study were protein kinases of the CMGC kinase group as well as some proteins of unknown function and proteins involved in energy metabolism, protein synthesis, and RNA metabolism. Interestingly, immunofluorescence microscopy using anti-phosphotyrosine antibodies suggests that there is a concentration of tyrosine-phosphorylated proteins associated with cytoskeletal structures (basal body and flagellum) and in the nucleolus of the parasite. This localization of tyrosine-phosphorylated proteins supports the idea that the function of signaling molecules is controlled by their precise location in
T. brucei
, a principle well known from higher eukaryotes.
Anthrax Protective Antigen Delivered bySalmonella entericaSerovar Typhi Ty21a Protects Mice from a Lethal Anthrax Spore Challenge
Authors: Osorio Manuel , Wu Yanping , Singh Sunil , Merkel Tod J. , Bhattacharyya Siba , Blake Milan S. , Kopecko Dennis J. 
Published: January 2009

ABSTRACTBacillus anthracis, the etiological agent of anthrax disease, is a proven weapon of bioterrorism. Currently, the only licensed vaccine against anthrax in the United States is AVA Biothrax, which, although efficacious, suffers from several limitations. This vaccine requires six injectable doses over 18 months to stimulate protective immunity, requires a cold chain for storage, and in many cases has been associated with adverse effects. In this study, we modified theB. anthracisprotective antigen (PA) gene for optimal expression and stability, linked it to an inducible promoter for maximal expression in the host, and fused it to the secretion signal of theEscherichia colialpha-hemolysin protein (HlyA) on a low-copy-number plasmid. This plasmid was introduced into the licensed typhoid vaccine strain,Salmonella entericaserovar Typhi strain Ty21a, and was found to be genetically stable. Immunization of mice with three vaccine doses elicited a strong PA-specific serum immunoglobulin G response with a geometric mean titer of 30,000 (range, 5,800 to 157,000) and lethal-toxin-neutralizing titers greater than 16,000. Vaccinated mice demonstrated 100% protection against a lethal intranasal challenge with aerosolized spores ofB. anthracis7702. The ultimate goal is a temperature-stable, safe, oral human vaccine against anthrax infection that can be self-administered in a few doses over a short period of time.
Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT
Authors: Nanatani Kei , Maloney Peter C. , Abe Keietsu 
Published: January 2009

ABSTRACTAspT, the aspartate:alanine antiporter ofTetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates ofl-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence ofl-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast,l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoesl-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit.
Identification of the Polyketide Synthase Involved in the Biosynthesis of the Surface-Exposed Lipooligosaccharides in Mycobacteria
Authors: Etienne Gilles , Malaga Wladimir , Laval Françoise , Lemassu Anne , Guilhot Christophe , Daffé Mamadou 
Published: January 2009

ABSTRACT

Lipooligosaccharides (LOS) are highly antigenic glycolipids produced by a number of
Mycobacterium
species, which include “
M. canettii
,” a member of the
M. tuberculosis
complex, and the opportunistic pathogens
M. marinum
and
M. kansasii
. The various LOS share a core composed of trehalose esterified by at least 1 mole of polymethyl-branched fatty acid (PMB-FA) and differ from one another by their oligosaccharide extensions. In this study, we identified a cluster of genes, MSMEG_4727 through MSMEG_4741, likely involved in the synthesis of LOS in
M. smegmatis
. Disruption of MSMEG_4727 (the ortholog of
pks5
of
M. tuberculosis
), which encodes a putative polyketide synthase, resulted in the concomitant abrogation of the production of both PMB-FA and LOS in the mutant strain. Complementation of the mutant with the wild-type gene fully restored the phenotype. We also showed that, in contrast to the case for “
M. canettii
” and
M. marinum
, LOS are located in deeper compartments of the cell envelope of
M. smegmatis
. The availability of two mycobacterial strains differing only in LOS production should help in defining the biological role(s) of this important glycolipid.
The Rv1712 Locus from
Mycobacterium tuberculosis
H37Rv Codes for a Functional CMP Kinase That Preferentially Phosphorylates dCMP
Authors: Thum Caroline , Schneider Cristopher Z. , Palma Mario S. , Santos Diógenes S. , Basso Luiz A. 
Published: January 2009

ABSTRACT

The
Mycobacterium tuberculosis cmk
gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that
M. tuberculosis
CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.
Properties of HflX, an Enigmatic Protein from
Escherichia coli
Authors: Dutta Dipak , Bandyopadhyay Kaustav , Datta Ajit Bikram , Sardesai Abhijit A. , Parrack Pradeep 
Published: January 2009

ABSTRACT

The
Escherichia coli
gene
hflX
was first identified as part of the
hflA
operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.
Expression and Mutational Analysis of the
glnB
Genomic Region in the Heterocyst-Forming Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Paz-Yepes Javier , Flores Enrique , Herrero Antonia 
Published: January 2009

ABSTRACT

In the filamentous, heterocyst-forming cyanobacterium
Anabaena
sp. strain PCC 7120, the
glnB
gene is expressed at considerable levels both in the presence and in the absence of combined nitrogen, although induction, influenced by NtcA, takes place upon combined-nitrogen deprivation likely localized to vegetative cells. In spite of extensive efforts, a derivative of PCC 7120 lacking a functional
glnB
gene could be obtained only with constructs that lead to overexpression of a downstream open reading frames (ORF), particularly all2318. Strain CSP10 [
glnB
all2318(Con)] exhibited growth rates similar to those of the wild type when it was using nitrate or ammonium, but its diazotrophic growth was impaired. However, it differentiated heterocysts with a time course and distribution pattern similar to those of the wild type, although with no cyanophycin-containing polar granules, and exhibited impaired nitrogenase activity under oxic conditions, but not under microoxic conditions. In the mutant, NtcA-dependent inducion of the
hetC
and
nifH
genes was unaltered, but induction of the
urtA
gene and urea transport activity were increased. Active uptake of nitrite was also increased and insensitive to the ammonium-promoted inhibition observed for the wild type. Thus, regulation of the nitrite transport activity requires the
glnB
gene product. In the presence of a wild-type
glnB
gene, neither inactivation nor overexpression of all2318 produced an apparent phenotype. Thus, in an otherwise wild-type background, the
glnB
gene appears to be essential for growth of strain PCC 7120. For growth with combined nitrogen but not for diazotrophic growth, the requirement for
glnB
can be overridden by increasing the expression of all2318 (and/or ORFs downstream of it).
Constant
c
10
Ring Stoichiometry in the
Escherichia coli
ATP Synthase Analyzed by Cross-Linking
Authors: Ballhausen Britta , Altendorf Karlheinz , Deckers-Hebestreit Gabriele 
Published: January 2009

ABSTRACT

The subunit
c
stoichiometry of
Escherichia coli
ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit
c
(
c
A21C/
c
M65C). Independent of the carbon source used for growth and independent of the presence of other F
o
F
1
subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.
Conjugative Transfer of the Integrative Conjugative Elements ICE
St1
and ICE
St3
from
Streptococcus thermophilus
Authors: Bellanger Xavier , Roberts Adam P. , Morel Catherine , Choulet Frédéric , Pavlovic Guillaume , Mullany Peter , Decaris Bernard , Guédon Gérard 
Published: January 2009

ABSTRACT

Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in
Streptococcus thermophilus
, ICE
St1
and ICE
St3
. Mitomycin C, a DNA-damaging agent, derepresses ICE
St3
conjugative transfer almost 25-fold. The ICE
St3
host range was determined using various members of the
Firmicutes
as recipients. Whereas numerous ICE
St3
transconjugants of
Streptococcus pyogenes
and
Enterococcus faecalis
were recovered, only one transconjugant of
Lactococcus lactis
was obtained. The newly incoming ICEs, except the one from
L. lactis
, are site-specifically integrated into the 3′ end of the
fda
gene and are still able to excise in these transconjugants. Furthermore, ICE
St3
was retransferred from
E. faecalis
to
S. thermophilus
. Recombinant plasmids carrying different parts of the ICE
St1
recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.
Different Biochemical Mechanisms Ensure Network-Wide Balancing of Reducing Equivalents in Microbial Metabolism
Authors: Fuhrer Tobias , Sauer Uwe 
Published: January 2009

ABSTRACTTo sustain growth, the catabolic formation of the redox equivalent NADPH must be balanced with the anabolic demand. The mechanisms that ensure such network-wide balancing, however, are presently not understood. Based on13C-detected intracellular fluxes, metabolite concentrations, and cofactor specificities for all relevant central metabolic enzymes, we have quantified catabolic NADPH production inAgrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, Paracoccus versutus, Pseudomonas fluorescens, Rhodobacter sphaeroides, Sinorhizobium meliloti, andZymomonas mobilis. For six species, the estimated NADPH production from glucose catabolism exceeded the requirements for biomass synthesis. Exceptions wereP. fluorescens, with balanced rates, andE. coli, with insufficient catabolic production, in which about one-third of the NADPH is supplied via the membrane-bound transhydrogenase PntAB.P. versutusandB. subtiliswere the only species that appear to rely on transhydrogenases for balancing NADPH overproduction during growth on glucose. In the other four species, the main but not exclusive redox-balancing mechanism appears to be the dual cofactor specificities of several catabolic enzymes and/or the existence of isoenzymes with distinct cofactor specificities, in particular glucose 6-phosphate dehydrogenase. An unexpected key finding for all species, exceptE. coliandB. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being NADP+dependent.
Role of Potassium Uptake Systems in
Sinorhizobium meliloti
Osmoadaptation and Symbiotic Performance
Authors: Domínguez-Ferreras Ana , Muñoz Socorro , Olivares José , Soto María J. , Sanjuán Juan 
Published: January 2009

ABSTRACT

Stimulation of potassium uptake is the most rapid response to an osmotic upshock in bacteria. This cation accumulates by a number of different transport systems whose importance has not been previously addressed for rhizobia. In silico analyses reveal the presence of genes encoding four possible potassium uptake systems in the genome of
Sinorhizobium meliloti
1021: Kup1, Kup2, Trk, and Kdp. The study of the relevance of these systems under a number of different growth conditions and in symbiosis showed that the integrity of Kup1 or Trk is essential for growth under laboratory conditions even in osmotically balanced media and the absence of both systems leads to a reduced infectivity and competitiveness of the bacteria in alfalfa roots. Trk is the main system involved in the accumulation of potassium after an osmotic upshift and the most important system for growth of
S. meliloti
under hyperosmotic conditions. The other three systems, especially Kup1, are also relevant during the osmotic adaptation of the cell, and the relative importance of the Kdp system increases at low potassium concentrations.