bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in
Saccharomyces cerevisiae
Authors: Zelle Rintze M. , Trueheart Josh , Harrison Jacob C. , Pronk Jack T. , van Maris Antonius J. A.
Published: June 2010

Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type
Saccharomyces cerevisiae
. Pyruvate carboxylase-negative (Pyc
−
)
S. cerevisiae
strains cannot grow on glucose unless media are supplemented with C
4
compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the
S. cerevisiae
PEPCK encoded by
PCK1
is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in
S. cerevisiae
. Pyc
−
S. cerevisiae
strains constitutively overexpressing the PEPCK either from
S. cerevisiae
or from
Actinobacillus succinogenes
did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h
−1
, one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C
4
dicarboxylic acids in
S. cerevisiae
.
Freshwater Suspended Sediments and Sewage Are Reservoirs for Enterotoxin-Positive
Clostridium perfringens
Authors: Mueller-Spitz Sabrina R. , Stewart Lisa B. , Klump J. Val , McLellan Sandra L.
Published: June 2010

The release of fecal pollution into surface waters may create environmental reservoirs of feces-derived microorganisms, including pathogens.
Clostridium perfringens
is a commonly used fecal indicator that represents a human pathogen. The pathogenicity of this bacterium is associated with its expression of multiple toxins; however, the prevalence of
C. perfringens
with various toxin genes in aquatic environments is not well characterized. In this study,
C. perfringens
spores were used to measure the distribution of fecal pollution associated with suspended sediments in the nearshore waters of Lake Michigan. Particle-associated
C. perfringens
levels were greatest adjacent to the Milwaukee harbor and diminished in the nearshore waters. Species-specific PCR and toxin gene profiles identified 174 isolates collected from the suspended sediments, surface water, and sewage influent as
C. perfringens
type A. Regardless of the isolation source, the beta2 and enterotoxin genes were common among isolates. The suspended sediments yielded the highest frequency of
cpe
-carrying
C. perfringens
(61%) compared to sewage (38%). Gene arrangement of enterotoxin was investigated using PCR to target known insertion sequences associated with this gene. Amplification products were detected in only 9 of 90 strains, which suggests there is greater variability in
cpe
gene arrangement than previously described. This work presents evidence that freshwater suspended sediments and sewage influent are reservoirs for potentially pathogenic
cpe
-carrying
C. perfringens
spores.
Detection of
Vibrio cholerae
O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay
Authors: Wang Duochun , Xu Xuebin , Deng Xiaoling , Chen Changyi , Li Baisheng , Tan Hailing , Wang Haibo , Tang Song , Qiu Haiyan , Chen Jingdiao , Ke Bixia , Ke Changwen , Kan Biao
Published: June 2010

Environmental waters are an important reservoir for
Vibrio cholerae
, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect
V. cholerae
O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 10
3
CFU/ml for detection of
V. cholerae
O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 10
5
CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the
V. cholerae
O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146;
P
< 0.01) but lower than that of real-time PCR (29.5%, 43/146;
P
< 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the
V. cholerae
concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of
V. cholerae
in environmental water samples.
Molecular and Genetic Analyses of the Putative
Proteus
O Antigen Gene Locus
Authors: Wang Quan , Torzewska Agnieszka , Ruan Xiaojuan , Wang Xiaoting , Rozalski Antoni , Shao Zhujun , Guo Xi , Zhou Haijian , Feng Lu , Wang Lei
Published: June 2010

Proteus
species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The
Proteus
O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of
Proteus
isolates. Even though most
Proteus
O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative
Proteus
O antigen locus and demonstrated this region's high degree of heterogeneity by comparing sequences of 40
Proteus
isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative
Proteus
O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally,
Proteus
-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68
Proteus
O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative
Proteus
O antigen locus, and we describe a novel molecular classification method for the identification of different
Proteus
serogroups.
Two Functionally Distinct Ciliates Dwelling in
Acropora
Corals in the South China Sea near Sanya, Hainan Province, China
Authors: Qiu Dajun , Huang Liangmin , Huang Hui , Yang Jianhui , Lin Senjie
Published: June 2010

We detected and characterized two distinct scuticociliate ciliates inside
Acropora
corals in the South China Sea. One, voraciously foraging on
Symbiodinium
, resembled the brown band disease of ciliates. The other, which is closely related to
Paranophrys magna
, grazed on detritus instead of
Symbiodinium
. These two ciliates may serve contrasting functions (competitor versus “cleaner”) in the coral-ciliate-
Symbiodinium
triangular relationship.
Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs
Authors: Warnes S. L. , Green S. M. , Michels H. T. , Keevil C. W.
Published: June 2010

The increasing incidence of nosocomial infections caused by glycopeptide-resistant enterococci is a global concern. Enterococcal species are also difficult to eradicate with existing cleaning regimens; they can survive for long periods on surfaces, thus contributing to cases of reinfection and spread of antibiotic-resistant strains. We have investigated the potential use of copper alloys as bactericidal surfaces. Clinical isolates of vancomycin-resistant
Enterococcus faecalis
and
Enterococcus faecium
were inoculated onto copper alloy and stainless steel surfaces. Samples were assessed for the presence of viable cells by conventional culture, detection of actively respiring cells, and assessment of cell membrane integrity. Both species survived for up to several weeks on stainless steel. However, no viable cells were detected on any alloys following exposure for 1 h at an inoculum concentration of ≤10
4
CFU/cm
2
. Analysis of genomic and plasmid DNA from bacterial cells recovered from metal surfaces indicates substantial disintegration of the DNA following exposure to copper surfaces that is not evident in cells recovered from stainless steel. The DNA fragmentation is so extensive, and coupled with the rapid cell death which occurs on copper surfaces, that it suggests that mutation is less likely to occur. It is therefore highly unlikely that genetic information can be transferred to receptive organisms recontaminating the same area. A combination of effective cleaning regimens and contact surfaces containing copper could be useful not only to prevent the spread of viable pathogenic enterococci but also to mitigate against the occurrence of potential resistance to copper, biocides, or antibiotics and the spread of genetic determinants of resistance to other species.
C Terminus of Nce102 Determines the Structure and Function of Microdomains in the Saccharomyces cerevisiae Plasma Membrane
Authors: Loibl Martin , Grossmann Guido , Stradalova Vendula , Klingl Andreas , Rachel Reinhard , Tanner Widmar , Malinsky Jan , Opekarová Miroslava
Published: June 2010

The plasma membrane of the yeast
Saccharomyces cerevisiae
contains stably distributed lateral domains of specific composition and structure, termed MCC (
m
embrane
c
ompartment of arginine permease
C
an1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an
NCE102
deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the
NCE102
close homolog
FHN1
(the previously uncharacterized
YGR131W
) as well as by distant
Schizosaccharomyces pombe
homolog
fhn1
(
SPBC1685.13
). We conclude that this mechanism of plasma membrane organization is conserved through the phylum
Ascomycota
. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.
Downregulation of an Entamoeba histolytica Rhomboid Protease Reveals Roles in Regulating Parasite Adhesion and Phagocytosis
Authors: Baxt Leigh A. , Rastew Elena , Bracha Rivka , Mirelman David , Singh Upinder
Published: June 2010

Entamoeba histolyticais a deep-branching eukaryotic pathogen. Rhomboid proteases are intramembrane serine proteases, which cleave transmembrane proteins in, or in close proximity to, their transmembrane domain. We have previously shown thatE. histolyticacontains a single functional rhomboid protease (EhROM1) and has unique substrate specificity. EhROM1 is present on the trophozoite surface and relocalizes to internal vesicles during erythrophagocytosis and to the base of the cap during surface receptor capping. In order to further examine the biological function of EhROM1 we downregulated EhROM1 expression by >95% by utilizing the epigenetic silencing mechanism of the G3 parasite strain. Despite the observation that EhROM1 relocalized to the cap during surface receptor capping, EhROM1 knockdown [ROM(KD)] parasites had no gross changes in cap formation or complement resistance. However, ROM(KD) parasites demonstrated decreased host cell adhesion, a result recapitulated by treatment of wild-type parasites with DCI, a serine protease inhibitor with activity against rhomboid proteases. The reduced adhesion phenotype of ROM(KD) parasites was noted exclusively with healthy cells, and not with apoptotic cells. Additionally, ROM(KD) parasites had decreased phagocytic ability with reduced ingestion of healthy cells, apoptotic cells, and rice starch. Decreased phagocytic ability is thus independent of the reduced adhesion phenotype, since phagocytosis of apoptotic cells was reduced despite normal adhesion levels. The defect in host cell adhesion was not explained by altered expression or localization of the heavy subunit of the Gal/GalNAc surface lectin. These results suggest no significant role of EhROM1 in complement resistance but unexpected roles in parasite adhesion and phagocytosis.
The Candida albicans ESCRT Pathway Makes Rim101-Dependent and -Independent Contributions to Pathogenesis
Authors: Wolf Julie M. , Johnson Diedre J. , Chmielewski David , Davis Dana A.
Published: June 2010

Candida albicans
is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environmental characteristics. To adapt to these different sites,
C. albicans
must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subsequent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the
vps27
Δ/Δ mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the
vps27
Δ/Δ strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for
C. albicans
virulence.
Cryptococcus neoformans Histone Acetyltransferase Gcn5 Regulates Fungal Adaptation to the Host
Authors: O'Meara Teresa R. , Hay Christie , Price Michael S. , Giles Steve , Alspaugh J. Andrew
Published: June 2010

Cryptococcus neoformans
is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses, we identified the
C. neoformans
Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of
Saccharomyces cerevisiae
. The
C. neoformans
gcn5
Δ mutant strain has defects in high-temperature growth and capsule attachment to the cell surface, in addition to increased sensitivity to FK506 and oxidative stress. Treatment of wild-type cells with the histone acetyltransferase inhibitor garcinol mimics cellular effects of the
gcn5
Δ
mutation. Gcn5 regulates the expression of many genes that are important in responding to the specific environmental conditions encountered by
C. neoformans
inside the host. Accordingly, the
gcn5
Δ
mutant is avirulent in animal models of cryptococcosis. Our study demonstrates the importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow
C. neoformans
to respond appropriately to the human host.
The Pre-mRNA Splicing Machinery of Trypanosomes: Complex or Simplified?
Authors: Günzl Arthur
Published: June 2010

Trypanosomatids are early-diverged, protistan parasites of which
Trypanosoma brucei
,
Trypanosoma cruzi
, and several species of
Leishmania
cause severe, often lethal diseases in humans. To better combat these parasites, their molecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL)
trans
splicing in
T. brucei
established a key difference between parasites and hosts. In SL
trans
splicing, the capped 5′-terminal region of the small nuclear SL RNA is fused onto the 5′ end of each mRNA. This process, in conjunction with polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA by providing the cap structure. The reaction is a two-step transesterification process analogous to intron removal by
cis
splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to ∼170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicing factors were identified by standard genome annotation because trypanosomatid amino acid sequences are among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterization of the splicing factor repertoire in
T. brucei
, achieved by tandem affinity purification of splicing complexes, by systematic analysis of proteins containing RNA recognition motifs, and by mining the genome database. In addition, recent findings about functional differences between trypanosome and human pre-mRNA splicing factors are discussed.
Uga3 and Uga35/Dal81 Transcription Factors Regulate
UGA4
Transcription in Response to γ-Aminobutyric Acid and Leucine
Authors: Cardillo Sabrina Beatriz , Bermúdez Moretti Mariana , Correa García Susana
Published: June 2010

The
Saccharomyces cerevisiae
UGA4
gene encodes a permease capable of importing γ-aminobutyric acid (GABA) and δ-aminolevulinic acid (ALA) into the cell. GABA-dependent induction of this permease requires at least two positive-acting proteins, the specific factor Uga3 and the pleiotropic factor Uga35/Dal81.
UGA4
is subjected to a very complex regulation, and its induction is affected by the presence of extracellular amino acids; this effect is mediated by the plasma membrane amino acid sensor SPS. Our results show that leucine affects
UGA4
induction and that the SPS sensor and the downstream effectors Stp1 and Stp2 participate in this regulation. Moreover, we found that the Uga3 and Uga35/Dal81 transcription factors bind to the
UGA4
promoter in a GABA-dependent manner and that this binding is impaired by the presence of leucine. We also found that the Leu3 transcription factor negatively regulates
UGA4
transcription, although this seems to be through an indirect mechanism.
Characterization of N-Linked Protein Glycosylation in
Helicobacter pullorum
Authors: Jervis Adrian J. , Langdon Rebecca , Hitchen Paul , Lawson Andrew J. , Wood Alison , Fothergill Joanne L. , Morris Howard R. , Dell Anne , Wren Brendan , Linton Dennis
Published: June 2010

The first bacterial N-linked glycosylation system was discovered in
Campylobacter jejuni
, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that
pglB
orthologues are present in a subset of species from the
Deltaproteobacteria
and
Epsilonproteobacteria
, including three
Helicobacter
species:
H. pullorum
,
H. canadensis
, and
H. winghamensis
. In contrast to
C. jejuni
, in which a single
pglB
gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these
Helicobacter
species contain two unrelated
pglB
genes (
pglB1
and
pglB2
), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the
H. pullorum
PglB1 protein, but not PglB2, was able to transfer
C. jejuni
N-linked glycan onto an acceptor protein in
Escherichia coli
. Analysis of the characterized
C. jejuni
N-glycosylation system with an
in vitro
oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to
H. pullorum
, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the −2 position of the N-glycosylation sequon, as for
C. jejuni
. Attempted insertional knockout mutagenesis of the
H. pullorum pglB2
gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied
C. jejuni
system.
Size Dependence of Protein Diffusion in the Cytoplasm of
Escherichia coli
Authors: Nenninger Anja , Mastroianni Giulia , Mullineaux Conrad W.
Published: June 2010

Diffusion in the bacterial cytoplasm is regarded as the primary method of intracellular protein movement and must play a major role in controlling the rates of cell processes. A number of recent studies have used green fluorescent protein (GFP) tagging and fluorescence microscopy to probe the movement and distribution of proteins in the bacterial cytoplasm. However, the dynamic behavior of indigenous proteins must be controlled by a complex mixture of specific interactions, combined with the basic physical constraints imposed by the viscosity and macromolecular crowding of the cytoplasm. These factors are difficult to unravel in studies with indigenous proteins. To what extent the addition of a GFP tag might affect the movement of a protein through the cytoplasm has also remained unknown. To resolve these problems, we have carried out a systematic study of the size dependence of protein diffusion coefficients in the
Escherichia coli
cytoplasm, using engineered GFP multimers (from 2 to 6 covalently linked GFP molecules). Diffusion coefficients were measured using confocal fluorescence recovery after photobleaching (FRAP). At least up to 110 kDa (four linked GFP molecules), the diffusion coefficient varies with size roughly as would be predicted from the Einstein-Stokes equation for a classical (Newtonian) fluid. Thus, protein diffusion coefficients are predictable over this range. GFP tagging of proteins has little impact on the diffusion coefficient over this size range and therefore need not significantly perturb protein movement. Two indigenous
E. coli
proteins were used to show that their specific interactions within the cell are the main controllers of the diffusion rate.
Functional Relationships between the AcrA Hairpin Tip Region and the TolC Aperture Tip Region for the Formation of the Bacterial Tripartite Efflux Pump AcrAB-TolC
Authors: Kim Hong-Man , Xu Yongbin , Lee Minho , Piao Shunfu , Sim Se-Hoon , Ha Nam-Chul , Lee Kangseok
Published: June 2010

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the α-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we providein vivofunctional data showing that both the length and the amino acid sequence of the α-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria.
Identification of a Receptor Subunit and Putative Ligand-Binding Residues Involved in the
Bacillus megaterium
QM B1551 Spore Germination Response to Glucose
Authors: Christie Graham , Götzke Hansjörg , Lowe Christopher R.
Published: June 2010

The molecular basis for the recognition of glucose as a germinant molecule by spores of
Bacillus megaterium
QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed
gerWB
) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.
Complete Nucleotide Sequence of TOL Plasmid pDK1 Provides Evidence for Evolutionary History of IncP-7 Catabolic Plasmids
Authors: Yano Hirokazu , Miyakoshi Masatoshi , Ohshima Kenshiro , Tabata Michiro , Nagata Yuji , Hattori Masahira , Tsuda Masataka
Published: June 2010

To understand the mechanisms for structural diversification ofPseudomonas-derived toluene-catabolic (TOL) plasmids, the complete sequence of a self-transmissible plasmid pDK1 with a size of 128,921 bp fromPseudomonasputidaHS1 was determined. Comparative analysis revealed that (i) pDK1 consisted of a 75.6-kb IncP-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pDK1 were highly similar to those of MOBHgroup of mobilizable plasmids, and (iii) the toluene-catabolic (xyl) gene clusters of pDK1 were derived through homologous recombination, transposition, and site-specific recombination from thexylgene clusters homologous to another TOL plasmid, pWW53. The minireplicons of pDK1 and its related IncP-7 plasmids, pWW53 and pCAR1, that contain replication and partition genes were maintained in all of sixPseudomonasstrains tested, but not in alpha- or betaproteobacterial strains. The recipient host range of conjugative transfer of pDK1 was, however, limited to twoPseudomonasstrains. These results indicate that IncP-7 plasmids are essentially narrow-host-range and self-transmissible plasmids that encode MOBHgroup-related transfer functions and that the host range of IncP-7-specified conjugative transfer was, unlike the situation in other well-known plasmids, narrower than that of its replication.
(p)ppGpp Inhibits Polynucleotide Phosphorylase from
Streptomyces
but Not from
Escherichia coli
and Increases the Stability of Bulk mRNA in
Streptomyces coelicolor
Authors: Gatewood Marcha L. , Jones George H.
Published: June 2010

ppGpp regulates gene expression in a variety of bacteria and in plants. We proposed previously that ppGpp or its precursor, pppGpp [referred to collectively as (p)ppGpp], or both might regulate the activity of the enzyme polynucleotide phosphorylase in
Streptomyces
species. We have examined the effects of (p)ppGpp on the polymerization and phosphorolysis activities of PNPase from
Streptomyces coelicolor
,
Streptomyces antibioticus
, and
Escherichia coli
. We have shown that (p)ppGpp inhibits the activities of both
Streptomyces
PNPases but not the
E. coli
enzyme. The inhibition kinetics for polymerization using the
Streptomyces
enzymes are of the mixed noncompetitive type, suggesting that (p)ppGpp binds to a region other than the active site of the enzyme. ppGpp also inhibited the phosphorolysis of a model RNA substrate derived from the
rpsO-pnp
operon of
S. coelicolor
. We have shown further that the chemical stability of mRNA increases during the stationary phase in
S. coelicolor
and that induction of a plasmid-borne copy of
relA
in a
relA
-null mutant increases the chemical stability of bulk mRNA as well. We speculate that the observed inhibition
in vitro
may reflect a role of ppGpp in the regulation of antibiotic production
in vivo
.
Differences in Cold Adaptation of
Bacillus subtilis
under Anaerobic and Aerobic Conditions
Authors: Beranová Jana , Mansilla María C. , de Mendoza Diego , Elhottová Dana , Konopásek Ivo
Published: June 2010

Bacillus subtilis
, which grows under aerobic conditions, employs fatty acid desaturase (Des) to fluidize its membrane when subjected to temperature downshift. Des requires molecular oxygen for its activity, and its expression is regulated by DesK-DesR, a two-component system. Transcription of
des
is induced by the temperature downshift and is decreased when membrane fluidity is restored.
B. subtilis
is also capable of anaerobic growth by nitrate or nitrite respiration. We studied the mechanism of cold adaptation in
B. subtilis
under anaerobic conditions that were predicted to inhibit Des activity. We found that in anaerobiosis, in contrast to aerobic growth, the induction of
des
expression after temperature downshift (from 37°C to 25°C) was not downregulated. However, the transfer from anaerobic to aerobic conditions rapidly restored the downregulation. Under both aerobic and anaerobic conditions, the induction of
des
expression was substantially reduced by the addition of external fluidizing oleic acid and was fully dependent on the DesK-DesR two-component regulatory system. Fatty acid analysis proved that there was no desaturation after
des
induction under anaerobic conditions despite the presence of high levels of the
des
protein product, which was shown by immunoblot analysis. The cold adaptation of
B. subtilis
in anaerobiosis is therefore mediated exclusively by the increased anteiso/iso ratio of branched-chain fatty acids and not by the temporarily increased level of unsaturated fatty acids that is typical under aerobic conditions. The degrees of membrane fluidization, as measured by diphenylhexatriene fluorescence anisotropy, were found to be similar under both aerobic and anaerobic conditions.
Regulation of Galactose Metabolism through the HisK:GalR Two-Component System in
Thermoanaerobacter tengcongensis
Authors: Qian Zhong , Wang Quanhui , Tong Wei , Zhou Chuanqi , Wang Qian , Liu Siqi
Published: June 2010

Thermoanaerobacter tengcongensis
could utilize galactose as a carbon source via the enzymes encoded by a novel
gal
operon, whose regulation mechanism has yet to be elucidated. We propose here that the
gal
operon in
T. tengcongensis
is regulated through a HisK:GalR two-component system. By using radioactive isotope assay and genetic analysis, we found that the kinase of this system, HisK, is phosphorylated by ATP, and the regulator, GalR, accepts a phosphoryl group during phosphorelay, in which the phosphoryl group at HisK-His-259 is transferred to GalR-Asp-56. Two-dimensional electrophoresis, followed by Western blotting, revealed that phosphorylation status of GalR is uniquely dependent on the galactose stimulus
in vivo
. Furthermore, DNA pulldown assays demonstrated that the phosphorylated GalR prefers binding to the operator DNA

O
2

, whereas the unphosphorylated GalR to

O
1

. A model of HisK:GalR is proposed to explain how galactose mediates the expression of the
gal
operon in
T. tengcongensis.
Rickettsia prowazekiiUses ansn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis
Authors: Frohlich Kyla M. , Roberts Rosemary A. W. , Housley Nicole A. , Audia Jonathon P.
Published: June 2010

Rickettsia prowazekiiis an obligate intracellular pathogen that possesses a small genome and a highly refined repertoire of biochemical pathways compared to those of free-living bacteria. Here we describe a novel biochemical pathway that relies on rickettsial transport of host cytosolic dihydroxyacetone phosphate (DHAP) and its subsequent conversion tosn-glycerol-3-phosphate (G3P) for synthesis of phospholipids. This rickettsial pathway compensates for the evolutionary loss of rickettsial glycolysis/gluconeogenesis, the typical endogenous source of G3P. One of the components of this pathway isR. prowazekiiopen reading frame RP442, which is annotated GpsA, a G3P dehydrogenase (G3PDH). Purified recombinant rickettsial GpsA was shown to specifically catalyze the conversion of DHAP to G3Pin vitro. The products of the GpsA assay were monitored spectrophotometrically, and the identity of the reaction product was verified by paper chromatography. In addition, heterologous expression of theR. prowazekii gpsAgene functioned to complement anEscherichia coli gpsAmutant. Furthermore,gpsAmRNA was detected inR. prowazekiipurified from hen egg yolk sacs, and G3PDH activity was assayable inR. prowazekiilysed-cell extracts. Together, these data strongly suggested thatR. prowazekiiencodes and synthesizes a functional GpsA enzyme, yetR. prowazekiiis unable to synthesize DHAP as a substrate for the GpsA enzymatic reaction. On the basis of the fact that intracellular organisms often avail themselves of resources in the host cell cytosol via the activity of novel carrier-mediated transport systems, we reasoned thatR. prowazekiitransports DHAP to supply substrate for GpsA. In support of this hypothesis, we show that purifiedR. prowazekiitransported and incorporated DHAP into phospholipids, thus implicating a role for GpsAin vivoas part of a novel rickettsial G3P acquisition pathway for phospholipid biosynthesis.
Compartmentalized Glucose Metabolism in
Pseudomonas putida
Is Controlled by the PtxS Repressor
Authors: Daddaoua Abdelali , Krell Tino , Alfonso Carlos , Morel Bertrand , Ramos Juan-Luis
Published: June 2010

Metabolic flux analysis revealed that in
Pseudomonas putida
KT2440 about 50% of glucose taken up by the cells is channeled through the 2-ketogluconate peripheral pathway. This pathway is characterized by being compartmentalized in the cells. In fact, initial metabolism of glucose to 2-ketogluconate takes place in the periplasm through a set of reactions catalyzed by glucose dehydrogenase and gluconate dehydrogenase to yield 2-ketogluconate. This metabolite is subsequently transported to the cytoplasm, where two reactions are carried out, giving rise to 6-phosphogluconate, which enters the Entner-Doudoroff pathway. The genes for the periplasmic and cytoplasmic set of reactions are clustered in the host chromosome and grouped within two independent operons that are under the control of the PtxS regulator, which also modulates its own synthesis. Here, we show that although the two catabolic operons are induced
in vivo
by glucose, ketogluconate, and 2-ketogluconate,
in vitro
we found that only 2-ketogluconate binds to the regulator with an apparent

K
D

(equilibrium dissociation constant) of 15 μM, as determined using isothermal titration calorimetry assays. PtxS is made of two domains, a helix-turn-helix DNA-binding domain located at the N terminus and a C-terminal domain that binds the effector. Differential scanning calorimetry assays revealed that PtxS unfolds via two events characterized by melting points of 48.1°C and 57.6°C and that, in the presence of 2-ketogluconate, the unfolding of the effector binding domain occurs at a higher temperature, providing further evidence for 2-ketogluconate-PtxS interactions. Purified PtxS is a dimer that binds to the target promoters with affinities in the range of 1 to 3 μM. Footprint analysis revealed that PtxS binds to an almost perfect palindrome that is present within the three promoters and whose consensus sequence is 5′-TGAAACCGGTTTCA-3′. This palindrome overlaps with the RNA polymerase binding site.
The
pdh
Operon Is Expressed in a Subpopulation of Stationary-Phase Bacteria and Is Important for Survival of Sugar-Starved
Streptococcus mutans
Authors: Busuioc Monica , Buttaro Bettina A. , Piggot Patrick J.
Published: June 2010

Streptococcus mutans
is a facultative member of the oral plaque and is associated with dental caries. It is able to survive long periods of sugar starvation. We show here that inactivation of
pdhD
, putatively encoding a subunit of the pyruvate dehydrogenase complex, impairs survival of both batch cultures and biofilms. We show that
pdhD
and the downstream genes
pdhA
,
pdhB
, and
pdhC
form an operon that is predominantly transcribed in stationary phase. Analysis with fluorescent reporters revealed a bimodal expression pattern for the
pdh
promoter, with less than 1% of stationary-phase populations expressing
pdh
. When it was first detected, after 1 day of sugar starvation in batch culture, expression was mostly in individual bacteria. At later times, expressing bacteria were often in chains. The lengths of the chains increased with time. We infer that the
pdh
-expressing subpopulation is able grow and divide and to persist for extended times in stationary phase.
Genome Sequence of the Milbemycin-Producing Bacterium
Streptomyces
bingchenggensis
Authors: Wang Xiang-Jing , Yan Yi-Jun , Zhang Bo , An Jing , Wang Ji-Jia , Tian Jun , Jiang Ling , Chen Yi-Hua , Huang Sheng-Xiong , Yin Min , Zhang Ji , Gao Ai-Li , Liu Chong-Xi , Zhu Zhao-Xiang , Xiang Wen-Sheng
Published: June 2010

Streptomyces
bingchenggensis
is a soil-dwelling bacterium producing the commercially important anthelmintic macrolide milbemycins. Besides milbemycins, the insecticidal polyether antibiotic nanchangmycin and some other antibiotics have also been isolated from this strain. Here we report the complete genome sequence of
S
.
bingchenggensis
. The availability of the genome sequence of
S
.
bingchenggensis
should enable us to understand the biosynthesis of these structurally intricate antibiotics better and facilitate rational improvement of this strain to increase their titers.
Electrophysiological studies of transgenic long QT type 1 and type 2 rabbits reveal genotype-specific differences in ventricular refractoriness and His conduction
Authors: Odening Katja E. , Kirk Malcolm , Brunner Michael , Ziv Ohad , Lorvidhaya Peem , Liu Gong Xin , Schofield Lorraine , Chaves Leonard , Peng Xuwen , Zehender Manfred , Choi Bum-Rak , Koren Gideon
Published: June 2010

We have generated transgenic rabbits lacking cardiac slow delayed-rectifier K+current [ IKs; long QT syndrome type 1 (LQT1)] or rapidly activating delayed-rectifier K+current [ IKr; long QT syndrome type 2 (LQT2)]. Rabbits with either genotype have prolonged action potential duration and QT intervals; however, only LQT2 rabbits develop atrioventricular (AV) blocks and polymorphic ventricular tachycardia. We therefore sought to characterize the genotype-specific differences in AV conduction and ventricular refractoriness in LQT1 and LQT2 rabbits. We carried out in vivo electrophysiological studies in LQT1, LQT2, and littermate control (LMC) rabbits at baseline, during isoproterenol infusion, and after a bolus of dofetilide and ex vivo optical mapping studies of the AV node/His-region at baseline and during dofetilide perfusion. Under isoflurane anesthesia, LQT2 rabbits developed infra-His blocks, decremental His conduction, and prolongation of the Wenckebach cycle length. In LQT1 rabbits, dofetilide altered the His morphology and slowed His conduction, resulting in intra-His block, and additionally prolonged the ventricular refractoriness, leading to pseudo-AV block . The ventricular effective refractory period (VERP) in right ventricular apex and base was significantly longer in LQT2 than LQT1 ( P < 0.05) or LMC ( P < 0.01), with a greater VERP dispersion in LQT2 than LQT1 rabbits. Isoproterenol reduced the VERP dispersion in LQT2 rabbits by shortening the VERP in the base more than in the apex but had no effect on VERP in LQT1. EPS and optical mapping experiments demonstrated genotype-specific differences in AV conduction and ventricular refractoriness. The occurrence of infra-His blocks in LQT2 rabbits under isoflurane and intra-His block in LQT1 rabbits after dofetilide suggest differential regional sensitivities of the rabbit His-Purkinje system to drugs blocking IKrand IKs.
Molecular mechanisms mediating preconditioning following chronic ischemia differ from those in classical second window
Authors: Depre Christophe , Park Ji Yeon , Shen You-Tang , Zhao Xin , Qiu Hongyu , Yan Lin , Tian Bin , Vatner Stephen F. , Vatner Dorothy E.
Published: June 2010

A major difference between experimental ischemic preconditioning (IPC), induced by brief ischemic episodes, and the clinical situation is that patients generally have repetitive episodes of ischemia. We used a swine model to examine differences in genes regulated by classical second-window IPC (SWOP) [two 10-min episodes of coronary artery occlusion (CAO) followed by 24 h reperfusion] compared with repetitive CAO/reperfusion (RCO), i.e., two 10-min CAO 12 h apart, and to repetitive coronary stenosis (RCS), six episodes of 90 min coronary stenosis 12 h apart ( n = 5/group). All three models reduced infarct size by 60–85%, which was mediated by nitric oxide in SWOP but not in the other two models. We employed microarray analyses to discover additional molecular pathways intrinsic to models of repetitive ischemia and different from classical SWOP. There was an 85% homology in gene response between the RCO and RCS models, whereas SWOP was qualitatively different. Both RCO and RCS, but not SWOP, showed downregulation of genes encoding proteins involved in oxidative metabolism and upregulation of genes involved in protein synthesis, unfolded protein response, autophagy, heat shock response, protein secretion, and an activation of the NF-κB signaling pathway. Therefore, the regulated genes mediating IPC with repetitive ischemia differ radically from SWOP both quantitatively and qualitatively, showing that a repetitive pattern of ischemia, rather than the difference between no-flow vs. low-flow ischemia, dictates the genomic response of the heart. These findings illustrate new cardioprotective mechanisms developed by repetitive IPC, which are potentially more relevant to patients with chronic ischemic heart disease, who are subjected to repetitive episodes of ischemia.
Chronic intermittent hypoxia in humans during 28 nights results in blood pressure elevation and increased muscle sympathetic nerve activity
Authors: Gilmartin G. S. , Lynch M. , Tamisier R. , Weiss J. W.
Published: June 2010

Chronic intermittent hypoxia (CIH) is thought to be responsible for the cardiovascular disease associated with obstructive sleep apnea (OSA). Increased sympathetic activation, altered vascular function, and inflammation are all putative mechanisms. We recently reported (Tamisier R, Gilmartin GS, Launois SH, Pepin JL, Nespoulet H, Thomas RJ, Levy P, Weiss JW. J Appl Physiol 107: 17–24, 2009) a new model of CIH in healthy humans that is associated with both increases in blood pressure and augmented peripheral chemosensitivity. We tested the hypothesis that exposure to CIH would also result in augmented muscle sympathetic nerve activity (MSNA) and altered vascular reactivity contributing to blood pressure elevation. We therefore exposed healthy subjects between the ages of 20 and 34 yr ( n = 7) to 9 h of nocturnal intermittent hypoxia for 28 consecutive nights. Cardiovascular and hemodynamic variables were recorded at three time points; MSNA was collected before and after exposure. Diastolic blood pressure (71 ± 1.3 vs. 74 ± 1.7 mmHg, P < 0.01), MSNA [9.94 ± 2.0 to 14.63 ± 1.5 bursts/min ( P < 0.05); 16.89 ± 3.2 to 26.97 ± 3.3 bursts/100 heartbeats (hb) ( P = 0.01)], and forearm vascular resistance (FVR) (35.3 ± 5.8 vs. 55.3 ± 6.5 mmHg·ml−1·min·100 g tissue, P = 0.01) all increased significantly after 4 wk of exposure. Forearm blood flow response following ischemia of 15 min (reactive hyperemia) fell below baseline values after 4 wk, following an initial increase after 2 wk of exposure. From these results we conclude that the increased blood pressure following prolonged exposure to CIH in healthy humans is associated with sympathetic activation and augmented FVR.
Effects of hydration and dehydration on blood rheology in sickle cell trait carriers during exercise
Authors: Tripette Julien , Loko Gylna , Samb Abdoulaye , Gogh Bertin Doubi , Sewade Estelle , Seck Djibril , Hue Olivier , Romana Marc , Diop Saliou , Diaw Mor , Brudey Karine , Bogui Pascal , Cissé Fallou , Hardy-Dessources Marie-Dominique , Connes Philippe
Published: June 2010

This study compared the hemorheological responses of a group of sickle cell trait (SCT) carriers with those of a control (Cont) group in response to 40 min of submaximal exercise (exercise intensity, 55% aerobic peak power) performed in two conditions: one with water offered ad libitum, i.e., the hydration (Hyd) condition, and one without water, i.e., the dehydration (Dehyd) condition. Blood and plasma viscosities, as well as red blood cell rigidity, were determined at rest, at the end of exercise, and at 2 h recovery with a cone plate viscometer at high shear rate and 37°C. The SCT and Cont groups lost 1 ± 0.7 and 1.6 ± 0.6 kg of body weight, respectively, in the Dehyd condition, indicating a significant effect of water deprivation compared with the Hyd condition, in which body weight remained unchanged. Plasma viscosity increased with exercise and returned to baseline during recovery independently of the group and condition. As previously demonstrated, resting blood viscosity was greater in the SCT carriers than in the Cont group. Blood viscosity increased by the end of exercise and returned to baseline at 2 h recovery in the Cont group in both conditions. The blood viscosity of SCT carriers did not change in response to exercise in the Dehyd condition and remained elevated at 2 h recovery. This extended hyperviscosity, in association with other biological changes induced by exercise, could be considered as a risk factor for exercise-related events in SCT carriers, similar to vasoocclusive crises, notably during the recovery. In contrast, the Hyd condition normalized the hyperviscosity and red blood cell rigidity of the SCT carriers, with blood viscosity values reaching the same lower values as those found in the Cont group during the recovery. Adequate hydration of SCT carriers should be strongly promoted to reduce the clinical risk associated with potential hyperviscosity complications.