bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Oxide as a Signaling Factor To Upregulate the Death-Specific Protein in a Marine Diatom,
Skeletonema costatum
, during Blockage of Electron Flow in Photosynthesis
Authors: Chung Chih-Ching , Hwang Sheng-Ping L. , Chang Jeng
Published: September 2008

To determine the physiological functions of a novel death-specific protein gene,
Skeletonema costatum DSP-1
(Sc
DSP-1
) in a marine diatom,
Skeletonema costatum
, the mRNA abundance of Sc
DSP-1
was measured in cultures subjected to light manipulation and treatments with various chemicals. When cells were transferred to a dim light intensity of 15 μmol m
−2
s
−1
, Sc
DSP-1
mRNA levels showed a transient increase of 1 to 17.2 μmol (mol 18S rRNA)
−1
in 60 h. Furthermore, treatments with the photoinhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-
p
-benzoquinone (DBMIB) resulted in high Sc
DSP-1
mRNA levels, which reached 943 and 72 μmol (mol 18S rRNA)
−1
, respectively. Treatment with the nitric oxide (NO) donor diethylamine nitric oxide also induced Sc
DSP-1
expression, and this inducible expression was inhibited by the NO scavenger hemoglobin. Additionally, the expression of Sc
DSP-1
mRNA elicited by DCMU and DBMIB was efficiently reduced when cultures were pretreated with the cell-penetrating NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. In contrast, treatment with another photoinhibitor, paraquat, had no effect on Sc
DSP-1
expression. Our results indicated that NO is the crucial secondary messenger which signals the expression of Sc
DSP-1
when electron flow between photosystem II and photosystem I is blocked in
S. costatum
cells. In addition, the discovery of a similar gene, Sc
DSP-2
, is briefly described.
Lactate and Amino Acid Catabolism in the Cheese-Ripening Yeast
Yarrowia lipolytica
Authors: Mansour S. , Beckerich J. M. , Bonnarme P.
Published: September 2008

The consumption of lactate and amino acids is very important for microbial development and/or aroma production during cheese ripening. A strain of
Yarrowia lipolytica
isolated from cheese was grown in a liquid medium containing lactate in the presence of a low (0.1×) or high (2×) concentration of amino acids. Our results show that there was a dramatic increase in the growth of
Y. lipolytica
in the medium containing a high amino acid concentration, but there was limited lactate consumption. Conversely, lactate was efficiently consumed in the medium containing a low concentration of amino acids after amino acid depletion was complete. These data suggest that the amino acids are used by
Y. lipolytica
as a main energy source, whereas lactate is consumed following amino acid depletion. Amino acid degradation was accompanied by ammonia production corresponding to a dramatic increase in the pH. The effect of adding amino acids to a
Y. lipolytica
culture grown on lactate was also investigated. Real-time quantitative PCR analyses were performed with specific primers for five genes involved in amino acid transport and catabolism, including an amino acid transporter gene (
GAP1
) and four aminotransferase genes (
ARO8
,
ARO9
,
BAT1
, and
BAT2
). The expression of three genes involved in lactate transport and catabolism was also studied. These genes included a lactate transporter gene (
JEN1
) and two lactate dehydrogenase genes (
CYB2
-
1
and
CYB2
-
2
). Our data showed that
GAP1
,
BAT2
,
BAT1
, and
ARO8
were maximally expressed after 15 to 30 min following addition of amino acids (
BAT2
was the most highly expressed gene), while the maximum expression of
JEN1
,
CYB2
-
1
, and
CYB2
-
2
was delayed (≥60 min).
Evidence for the Role of Calcineurin in Morphogenesis and Calcium Homeostasis during Mycelium-to-Yeast Dimorphism of
Paracoccidioides brasiliensis
Authors: Campos Claudia B. L. , Di Benedette Joao Paulo T. , Morais Flavia V. , Ovalle Rafael , Nobrega Marina P.
Published: September 2008

Paracoccidioides brasiliensis
is a dimorphic fungus that causes paracoccidioidomycosis, the most prevalent human deep mycosis in Latin America. The dimorphic transition from mycelium to yeast (M-Y) is triggered by a temperature shift from 25°C to 37°C and is critical for pathogenicity. Intracellular Ca
2+
levels increased in hyphae immediately after temperature-induced dimorphism. The chelation of Ca
2+
with extracellular (EGTA) or intracellular (BAPTA) calcium chelators inhibited temperature-induced dimorphism, whereas the addition of extracellular Ca
2+
accelerated dimorphism. The calcineurin inhibitor cyclosporine A (CsA), but not tacrolimus (FK506), effectively decreased cell growth, halted the M-Y transition that is associated with virulence, and caused aberrant growth morphologies for all forms of
P. brasiliensis
. The difference between CsA and FK506 was ascribed by the higher levels of cyclophilins contrasted to FKBPs, the intracellular drug targets required for calcineurin suppression. Chronic exposure to CsA abolished intracellular Ca
2+
homeostasis and decreased mRNA transcription of the
CCH1
gene for the plasma membrane Ca
2+
channel in yeast-form cells. CsA had no detectable effect on multidrug resistance efflux pumps, while the effect of FK506 on rhodamine excretion was not correlated with the transition to yeast form. In this study, we present evidence that Ca
2+
/calmodulin-dependent phosphatase calcineurin controls hyphal and yeast morphology, M-Y dimorphism, growth, and Ca
2+
homeostasis in
P. brasiliensis
and that CsA is an effective chemical block for thermodimorphism in this organism. The effects of calcineurin inhibitors on
P. brasiliensis
reinforce the therapeutic potential of these drugs in a combinatory approach with antifungal drugs to treat endemic paracoccidioidomycosis.
Structural and Functional Association of
Trypanosoma brucei
MIX Protein with Cytochrome
c
Oxidase Complex
Authors: Zíková Alena , Panigrahi Aswini K. , Uboldi Alessandro D. , Dalley Rachel A. , Handman Emanuela , Stuart Kenneth
Published: September 2008

A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in
Leishmania major
. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of
Trypanosoma brucei
, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for
Leishmania
. The loss of MIX also had a major effect on cytochrome
c
oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome
c
oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in
T. brucei
.
A Signaling-Regulated, Short-Chain Dehydrogenase of
Stagonospora nodorum
Regulates Asexual Development
Authors: Tan Kar-Chun , Heazlewood Joshua L. , Millar A. Harvey , Thomson Gordon , Oliver Richard P. , Solomon Peter S.
Published: September 2008

The fungus
Stagonospora nodorum
is a causal agent of leaf and glume blotch disease of wheat. It has been previously shown that inactivation of heterotrimeric G protein signaling in
Stagonospora nodorum
caused development defects and reduced pathogenicity [P. S. Solomon et al., Mol. Plant-Microbe Interact. 17:456-466, 2004]. In this study, we sought to identify targets of the signaling pathway that may have contributed to phenotypic defects of the signaling mutants. A comparative analysis of
Stagonospora nodorum
wild-type and Gα-defective mutant (
gna1
) intracellular proteomes was performed via two-dimensional polyacrylamide gel electrophoresis. Several proteins showed significantly altered abundances when comparing the two strains. One such protein, the short-chain dehydrogenase Sch1, was 18-fold less abundant in the
gna1
strain, implying that it is positively regulated by Gα signaling. Gene expression and transcriptional enhanced green fluorescent protein fusion analyses of
Sch1
indicates strong expression during asexual development. Mutant strains of
Stagonospora nodorum
lacking
Sch1
demonstrated poor growth on minimal media and exhibited a significant reduction in asexual sporulation on all growth media examined. Detailed histological experiments on
sch1
pycnidia revealed that the gene is required for the differentiation of the subparietal layers of asexual pycnidia resulting in a significant reduction in both pycnidiospore size and numbers.
Cytological Characterization of YpsB, a Novel Component of the
Bacillus subtilis
Divisome
Authors: Tavares José Roberto , de Souza Robson F. , Meira Guilherme Louzada Silva , Gueiros-Filho Frederico J.
Published: September 2008

Cell division in bacteria is carried out by an elaborate molecular machine composed of more than a dozen proteins and known as the divisome. Here we describe the characterization of a new divisome protein in
Bacillus subtilis
called YpsB. Sequence comparisons and phylogentic analysis demonstrated that YpsB is a paralog of the division site selection protein DivIVA. YpsB is present in several gram-positive bacteria and likely originated from the duplication of a DivIVA-like gene in the last common ancestor of bacteria of the orders
Bacillales
and
Lactobacillales
. We used green fluorescent protein microscopy to determine that YpsB localizes to the divisome. Similarly to that for DivIVA, the recruitment of YpsB to the divisome requires late division proteins and occurs significantly after Z-ring formation. In contrast to DivIVA, however, YpsB is not retained at the newly formed cell poles after septation. Deletion analysis suggests that the N terminus of YpsB is required to target the protein to the divisome. The high similarity between the N termini of YpsB and DivIVA suggests that the same region is involved in the targeting of DivIVA. YpsB is not essential for septum formation and does not appear to play a role in septum positioning. However, a
ypsB
deletion has a synthetic effect when combined with a mutation in the cell division gene
ftsA
. Thus, we conclude that YpsB is a novel
B. subtilis
cell division protein whose function has diverged from that of its paralog DivIVA.
The Chromate-Inducible
chrBACF
Operon from the Transposable Element Tn
OtChr
Confers Resistance to Chromium(VI) and Superoxide
Authors: Branco Rita , Chung Ana Paula , Johnston Tatiana , Gurel Volkan , Morais Paula , Zhitkovich Anatoly
Published: September 2008

Large-scale industrial use of chromium(VI) has resulted in widespread contamination with carcinogenic chromium(VI). The abilities of microorganisms to survive in these environments and to detoxify chromate require the presence of specific resistance systems. Here we report identification of the transposon-located (Tn
OtChr
) chromate resistance genes from the highly tolerant strain
Ochrobactrum tritici
5bvl1 surviving chromate concentrations of >50 mM. The 7,189-bp-long Tn
OtChr
of the mixed Tn
21
/Tn
3
transposon subfamily contains a group of
chrB
,
chrA
,
chrC
, and
chrF
genes situated between divergently transcribed resolvase and transposase genes. The
chrB
and
chrA
genes, but not
chrF
or
chrC
, were essential for establishment of high resistance in chromium-sensitive
O. tritici
. The
chr
promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of
chr
expression. Induction of the
chr
operon suppressed accumulation of cellular Cr through the activity of a chromate efflux pump encoded by
chrA
. Expression of
chrB
,
chrC
, or
chrF
in an
Escherichia coli sodA sodB
double mutant restored its aerobic growth in minimal medium and conferred resistance to superoxide-generating agents menadione and paraquat. Nitroblue tetrazolium staining on native gels showed that ChrC protein had superoxide dismutase activity. Tn
OtChr
appears to represent a mobile genetic system for the distribution of the chromate-regulated resistance operon. The presence of three genes protecting against superoxide toxicity should provide an additional survival advantage to Tn
OtChr
-containing cells in the environments with multiple redox-active contaminants.
A Functional Phenylacetic Acid Catabolic Pathway Is Required for Full Pathogenicity of
Burkholderia cenocepacia
in the
Caenorhabditis elegans
Host Model
Authors: Law Robyn J. , Hamlin Jason N. R. , Sivro Aida , McCorrister Stuart J. , Cardama Georgina A. , Cardona Silvia T.
Published: September 2008

Burkholderia cenocepacia
is a member of the
Burkholderia cepacia
complex, a group of metabolically versatile bacteria that have emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Previously a screen of transposon mutants in a rat pulmonary infection model identified an attenuated mutant with an insertion in
paaE
, a gene related to the phenylacetic acid (PA) catabolic pathway. In this study, we characterized gene clusters involved in the PA degradation pathway of
B. cenocepacia
K56-2 in relation to its pathogenicity in the
Caenorhabditis elegans
model of infection. We demonstrated that targeted-insertion mutagenesis of
paaA
and
paaE
, which encode part of the putative PA-coenzyme A (CoA) ring hydroxylation system,
paaZ
, coding for a putative ring opening enzyme, and
paaF
, encoding part of the putative beta-oxidation system, severely reduces growth on PA as a sole carbon source.
paaA
and
paaE
insertional mutants were attenuated for virulence, and expression of
paaE
in
trans
restored pathogenicity of the
paaE
mutant to wild-type levels. Interruption of
paaZ
and
paaF
slightly increased virulence. Using gene interference by ingested double-stranded RNA, we showed that the attenuated phenotype of the
paaA
and
paaE
mutants is dependent on a functional p38 mitogen-activated protein kinase pathway in
C. elegans
. Taken together, our results demonstrate that
B. cenocepacia
possesses a functional PA degradation pathway and that the putative PA-CoA ring hydroxylation system is required for full pathogenicity in
C. elegans
.
The Bile Response Repressor BreR Regulates Expression of the
Vibrio cholerae breAB
Efflux System Operon
Authors: Cerda-Maira Francisca A. , Ringelberg Carol S. , Taylor Ronald K.
Published: September 2008

Enteric pathogens have developed several resistance mechanisms to survive the antimicrobial action of bile. We investigated the transcriptional profile of
Vibrio cholerae
O1 El Tor strain C6706 under virulence gene-inducing conditions in the presence and absence of bile. Microarray analysis revealed that the expression of 119 genes was affected by bile. The mRNA levels of genes encoding proteins involved in transport were increased in the presence of bile, whereas the mRNA levels of genes encoding proteins involved in pathogenesis and chemotaxis were decreased. This study identified genes encoding transcriptional regulators from the TetR family (
vexR
and
breR
) and multidrug efflux pumps from the resistance-nodulation-cell division superfamily (
vexB
and
vexD
[herein renamed
breB
]) that were induced in response to bile. Further analysis regarding
vexAB
and
breAB
expression in the presence of various antimicrobial compounds established that
vexAB
was induced in the presence of bile, sodium dodecyl sulfate, or novobiocin and that the induction of
breAB
was specific to bile. BreR is a direct repressor of the
breAB
promoter and is able to regulate its own expression, as demonstrated by transcriptional and electrophoretic mobility shift assays (EMSA). The expression of
breR
and
breAB
is induced in the presence of the bile salts cholate, deoxycholate, and chenodeoxycholate, and EMSA showed that deoxycholate is able to abolish the formation of BreR-P

breR

complexes. We propose that deoxycholate is able to interact with BreR and induce a conformational change that interferes with the DNA binding ability of BreR, resulting in
breAB
and
breR
expression. These results provide new insight into a transcriptional regulator and a transport system that likely play essential roles in the ability of
V. cholerae
to resist the action of bile in the host.
SpoT-Triggered Stringent Response Controls
usp
Gene Expression in
Pseudomonas aeruginosa
Authors: Boes Nelli , Schreiber Kerstin , Schobert Max
Published: September 2008

The universal stress proteins (Usps) UspK (PA3309) and UspN (PA4352) of
Pseudomonas aeruginosa
are essential for surviving specific anaerobic energy stress conditions such as pyruvate fermentation and anaerobic stationary phase. Expression of the respective genes is under the control of the oxygen-sensing regulator Anr. In this study we investigated the regulation of
uspN
and three additional
P. aeruginosa usp
genes:
uspL
(PA1789),
uspM
(PA4328), and
uspO
(PA5027). Anr induces expression of these genes in response to anaerobic conditions. Using promoter-
lacZ
fusions, we showed that P

uspL

-
lacZ
, P

uspM

-
lacZ
, and P

uspO

-
lacZ
were also induced in stationary phase as described for P

uspN

-
lacZ
. However, stationary phase gene expression was abolished in the
P. aeruginosa
triple mutant Δ
anr
Δ
relA
Δ
spoT
. The
relA
and
spoT
genes encode the regulatory components of the stringent response. We determined pppGpp and ppGpp levels using a thin-layer chromatography approach and detected the accumulation of ppGpp in the wild type and the Δ
relA
mutant in stationary phase, indicating a SpoT-derived control of ppGpp accumulation. Additional investigation of stationary phase in LB medium revealed that alkaline pH values are involved in the regulatory process of ppGpp accumulation.
Lsr2 of
Mycobacterium
Represents a Novel Class of H-NS-Like Proteins
Authors: Gordon Blair R. G. , Imperial Robin , Wang Linru , Navarre William Wiley , Liu Jun
Published: September 2008

Lsr2 is a small, basic protein present in
Mycobacterium
and related actinomycetes. Our previous in vitro biochemical studies showed that Lsr2 is a DNA-bridging protein, a property shared by H-NS-like proteins in gram-negative bacteria. Here we present in vivo evidence based on genetic complementation experiments that Lsr2 is a functional analog of H-NS, the first such protein identified in gram-positive bacteria. We show that
lsr2
can complement the phenotypes related to
hns
mutations in
Escherichia coli
, including β-glucoside utilization, mucoidy, motility, and hemolytic activity. We also show that Lsr2 binds specifically to H-NS-regulated genes and the repression of
hlyE
by Lsr2 can be partially eliminated by overexpression of
slyA
, suggesting that the molecular mechanisms of Lsr2 repression and depression are similar to those of H-NS. The functional equivalence of these two proteins is further supported by the ability of
hns
to complement the
lsr2
phenotype in
Mycobacterium smegmatis
. Taken together, our results demonstrate unequivocally that Lsr2 is an H-NS-like protein.
PqsE Functions Independently of PqsR-PseudomonasQuinolone Signal and Enhances therhlQuorum-Sensing System
Authors: Farrow John M. , Sund Zoe M. , Ellison Matthew L. , Wade Dana S. , Coleman James P. , Pesci Everett C.
Published: September 2008

Pseudomonas aeruginosais an opportunistic pathogen that causes both acute and chronic infections in immunocompromised individuals. This gram-negative bacterium produces a battery of virulence factors that allow it to infect and survive in many different hostile environments. The control of many of these virulence factors falls under the influence of one of threeP. aeruginosacell-to-cell signaling systems. The focus of this study, the quinolone signaling system, functions through thePseudomonasquinolone signal (PQS), previously identified as 2-heptyl-3-hydroxy-4-quinolone. This signal binds to and activates the LysR-type transcriptional regulator PqsR (also known as MvfR), which in turn induces the expression of thepqsABCDEoperon. The first four genes of this operon are required for PQS synthesis, but the fifth gene,pqsE, is not. The function of thepqsEgene is not known, but it is required for the production of multiple PQS-controlled virulence factors and for virulence in multiple models of infection. In this report, we show that PqsE can activate PQS-controlled genes in the absence of PqsR and PQS. Our data also suggest that the regulatory activity of PqsE requires RhlR and indicate that apqsEmutant can be complemented for pyocyanin production by a large excess of exogenousN-butyryl homoserine lactone (C4-HSL). Finally, we show that PqsE enhances the ability ofEscherichia coliexpressing RhlR to respond to C4-HSL. Overall, our data lead us to conclude that PqsE functions as a regulator that is independent of PqsR and PQS but dependent on therhlquorum-sensing system.
Dual Role of FtsH in Regulating Lipopolysaccharide Biosynthesis in
Escherichia coli
Authors: Katz Chen , Ron Eliora Z.
Published: September 2008

In
Escherichia coli
, FtsH (HflB) is a membrane-bound, ATP-dependent metalloendoprotease belonging to the AAA family (ATPases associated with diverse cellular activities). FtsH has a limited spectrum of known substrates, including the transcriptional activator σ
32
. FtsH is the only known
E. coli
protease that is essential, as it regulates the concentration of LpxC, which carries out the first committed step in the synthesis of lipid A. Here we identify a new FtsH substrate—3-deoxy-
d
-
manno
-octulosonate (KDO) transferase—which carries out the attachment of two KDO residues to the lipid A precursor (lipid IV
A
) to form the minimal essential structure of the lipopolysaccharide (LPS) (KDO
2
-lipid A). Thus, FtsH regulates the concentration of the lipid moiety of LPS (lipid A) as well as the sugar moiety (KDO-based core oligosaccharides), ensuring a balanced synthesis of LPS.
A Novel Mutator of
Escherichia coli
Carrying a Defect in the
dgt
Gene, Encoding a dGTP Triphosphohydrolase
Authors: Gawel Damian , Hamilton Michael D. , Schaaper Roel M.
Published: September 2008

A novel mutator locus in
Escherichia coli
was identified from a collection of random transposon insertion mutants. Several mutators in this collection were found to have an insertion in the
dgt
gene, encoding a previously characterized dGTP triphosphohydrolase. The mutator activity of the
dgt
mutants displays an unusual specificity. Among the six possible base pair substitutions in a
lacZ
reversion system, the G·C→C·G transversion and A·T→G·C transition are strongly enhanced (10- to 50-fold), while a modest effect (two- to threefold) is also observed for the G·C→A·T transition. Interestingly, a two- to threefold reduction in mutant frequency (antimutator effect) is observed for the G·C→T·A transversion. In the absence of DNA mismatch repair (
mutL
) some of these effects are reduced or abolished, while other effects remain unchanged. Analysis of these effects, combined with the DNA sequence contexts in which the reversions take place, suggests that alterations of the dGTP pools as well as alterations in the level of some modified dNTP derivatives could affect the fidelity of in vivo DNA replication and, hence, account for the overall mutator effects.
Novel Proteins That Modulate Type IV Pilus Retraction Dynamics in
Pseudomonas aeruginosa
Authors: Asikyan Miranda L. , Kus Julianne V. , Burrows Lori L.
Published: September 2008

Pseudomonas aeruginosa
uses type IV pili to colonize various materials and for surface-associated twitching motility. We previously identified five phylogenetically distinct alleles of
pilA
in
P. aeruginosa
, four of which occur in genetic cassettes with specific accessory genes (J. V. Kus, E. Tullis, D. G. Cvitkovitch, and L. L. Burrows, Microbiology 150:1315-1326, 2004). Each of the five pilin alleles, with and without its associated pilin accessory gene, was used to complement a group II PAO1
pilA
mutant. Expression of group I or IV
pilA
genes restored twitching motility to the same extent as the PAO1 group II pilin. In contrast, poor twitching resulted from complementation with group III or group V
pilA
genes but increased significantly when the cognate
tfpY
or
tfpZ
accessory genes were cointroduced. The enhanced motility was linked to an increase in recoverable surface pili and not to alterations in total pilin pools. Expression of the group III or V pilins in a PAO1
pilA-pilT
double mutant yielded large amounts of surface pili, regardless of the presence of the accessory genes. Therefore, poor piliation in the absence of the TfpY and TfpZ accessory proteins results from a net increase in PilT-mediated retraction. Similar phenotypes were observed for
tfpY
single and
tfpY-pilT
double knockout mutants of group III strain PA14. A PilA
V
-TfpY chimera produced few surface pili, showing that the accessory proteins are specific for their cognate pilin. The genetic linkage between specific pilin and accessory genes may be evolutionarily conserved because the accessory proteins increase pilus expression on the cell surface, thereby enhancing function.
Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603Clostridium perfringensEnterotoxin Plasmid
Authors: Miyamoto Kazuaki , Li Jihong , Sayeed Sameera , Akimoto Shigeru , McClane Bruce A.
Published: September 2008

Clostridium perfringenstype B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, butetxgenetics have been poorly studied in type B isolates. This study reports the first sequencing of anyetxplasmid, i.e., pCP8533etx, from type B strain NCTC8533. Thisetxplasmid is 64.7 kb, carriestcpconjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-likeetxplasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive,cpe-negative type D isolates, while other type D isolates carry differentetxplasmids. Sequences upstream of theetxgene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-likeetxplasmid. However, nearly all type B and D isolates have anetxlocus with an upstream IS1151, and thoseetxloci typically reside near adcmORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin oretxgenes, respectively, onto a common progenitor plasmid.
Ethanolamine Activates a Sensor Histidine Kinase Regulating Its Utilization in
Enterococcus faecalis
Authors: Del Papa María Florencia , Perego Marta
Published: September 2008

Enterococcus faecalis
is a gram-positive commensal bacterium of the human intestinal tract. Its opportunistic pathogenicity has been enhanced by the acquisition of multiple antibiotic resistances, making the treatment of enterococcal infections an increasingly difficult problem. The extraordinary capacity of this organism to colonize and survive in a wide variety of ecological niches is attributable, at least in part, to signal transduction pathways mediated by two-component systems (TCS). Here, the ability of
E. faecalis
to utilize ethanolamine as the sole carbon source is shown to be dependent upon the RR-HK17 (EF1633-EF1632) TCS. Ethanolamine is an abundant compound in the human intestine, and thus, the ability of bacteria to utilize it as a source of carbon and nitrogen may provide an advantage for survival and colonization. Growth of
E. faecalis
in a synthetic medium with ethanolamine was abolished in the response regulator RR17 mutant strain. Transcription of the response regulator gene was induced by the presence of ethanolamine. Ethanolamine induced a 15-fold increase in the rate of autophosphorylation in vitro of the HK17 sensor histidine kinase, indicating that this is the ligand recognized by the sensor domain of the kinase. These results assign a role to the RR-HK17 TCS as coordinator of the enterococcal response to specific nutritional conditions existing at the site of bacterial invasion, the intestinal tract of an animal host.
PilF Is an Outer Membrane Lipoprotein Required for Multimerization and Localization of thePseudomonas aeruginosaType IV Pilus Secretin
Authors: Koo Jason , Tammam Stephanie , Ku Shao-Yang , Sampaleanu Liliana M. , Burrows Lori L. , Howell P. Lynne
Published: September 2008

Type IV pili (T4P) are retractile appendages that contribute to the virulence of bacterial pathogens. PilF is aPseudomonas aeruginosalipoprotein that is essential for T4P biogenesis. Phenotypic characterization of apilFmutant confirmed that T4P-mediated functions are abrogated: T4P were no longer present on the cell surface, twitching motility was abolished, and the mutant was resistant to infection by T4P retraction-dependent bacteriophage. The results of cellular fractionation studies indicated that PilF is the outer membrane pilotin required for the localization and multimerization of the secretin, PilQ. Mutation of the putative PilF lipidation site untethered the protein from the outer membrane, causing secretin assembly in both inner and outer membranes. T4P-mediated twitching motility and bacteriophage susceptibility were moderately decreased in the lipidation site mutant, while cell surface piliation was substantially reduced. The tethering of PilF to the outer membrane promotes the correct localization of PilQ and appears to be required for the formation of stable T4P. Our 2.0-Å structure of PilF revealed a superhelical arrangement of six tetratricopeptide protein-protein interaction motifs that may mediate the contacts with PilQ during secretin assembly. An alignment of pseudomonad PilF sequences revealed three highly conserved surfaces that may be involved in PilF function.
Identification of Genes Encoding the Folate- and Thiamine-Binding Membrane Proteins in Firmicutes
Authors: Eudes Aymerick , Erkens Guus B. , Slotboom Dirk J. , Rodionov Dmitry A. , Naponelli Valeria , Hanson Andrew D.
Published: September 2008

Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the
Lactobacillus casei
genome, expressed in
Lactococcus lactis
, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most
thiT
genes are preceded by a thiamine riboswitch.
The Human Immunodeficiency Virus Type 1 Envelope Confers Higher Rates of Replicative Fitness to Perinatally Transmitted Viruses than to Nontransmitted Viruses
Authors: Kong Xiaohong , West John T. , Zhang Hong , Shea Danielle M. , M'soka Tendai J. , Wood Charles
Published: September 2008

Selection of a minor viral genotype during perinatal transmission of human Immunodeficiency virus type 1 (HIV-1) has been observed, but there is a lack of information on the correlation of the restrictive transmission with biological properties of the virus, such as replicative fitness. Recombinant viruses expressing the enhanced green fluorescent protein or the
Discosoma
sp. red fluorescent (DsRed2) protein carrying the V1 to V5 regions of
env
from seven mother-infant pairs (MIPs) infected by subtype C HIV-1 were constructed, and competition assays were carried out to compare the fitness between the transmitted and nontransmitted viruses. Flow cytometry was used to quantify the frequency of infected cells, and the replicative fitness was determined based on a calculation that takes into account replication of competing viruses in a single infection versus dual infections. Transmitted viruses from five MIPs with the mothers chronically infected showed a restrictive
env
genotype, and all the recombinant viruses carrying the infants' Env had higher replicative fitness than those carrying the Env from the mothers. This growth fitness is lineage specific and can be observed only within the same MIP. In contrast, in two MIPs where the mothers had undergone recent acute infection, the viral Env sequences were similar between the mothers and infants and showed no further restriction in quasispecies during perinatal transmission. The recombinant viruses carrying the Env from the infants' viruses also showed replication fitness similar to those carrying the mothers' Env proteins. Our results suggest that newly transmitted viruses from chronically infected mothers have been selected to have higher replicative fitness to favor transmission, and this advantage is conferred by the V1 to V5 region of Env of the transmitted viruses. This finding has important implications for vaccine design or development of strategies to prevent HIV-1 transmission.
Entry from the Cell Surface of Severe Acute Respiratory Syndrome Coronavirus with Cleaved S Protein as Revealed by Pseudotype Virus Bearing Cleaved S Protein
Authors: Watanabe Rie , Matsuyama Shutoku , Shirato Kazuya , Maejima Masami , Fukushi Shuetsu , Morikawa Shigeru , Taguchi Fumihiro
Published: September 2008

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is known to take an endosomal pathway for cell entry; however, it is thought to enter directly from the cell surface when a receptor-bound virion spike (S) protein is affected by trypsin, which induces cleavage of the S protein and activates its fusion potential. This suggests that SARS-CoV bearing a cleaved form of the S protein can enter cells directly from the cell surface without trypsin treatment. To explore this possibility, we introduced a furin-like cleavage sequence in the S protein at amino acids 798 to 801 and found that the mutated S protein was cleaved and induced cell fusion without trypsin treatment when expressed on the cell surface. Furthermore, a pseudotype virus bearing a cleaved S protein was revealed to infect cells in the presence of a lysosomotropic agent as well as a protease inhibitor, both of which are known to block SARS-CoV infection via an endosome, whereas the infection of pseudotypes with an uncleaved, wild-type S protein was blocked by these agents. A heptad repeat peptide, derived from a SARS-CoV S protein that is known to efficiently block infections from the cell surface, blocked the infection by a pseudotype with a cleaved S protein but not that with an uncleaved S protein. Those results indicate that SARS-CoV with a cleaved S protein is able to enter cells directly from the cell surface and agree with the previous observation of the protease-mediated cell surface entry of SARS-CoV.
Multiple Virus Lineages Sharing Recent Common Ancestry Were Associated with a Large Rift Valley Fever Outbreak among Livestock in Kenya during 2006-2007
Authors: Bird Brian H. , Githinji Jane W. K. , Macharia Joseph M. , Kasiiti Jacqueline L. , Muriithi Rees M. , Gacheru Stephen G. , Musaa Joseph O. , Towner Jonathan S. , Reeder Serena A. , Oliver Jennifer B. , Stevens Thomas L. , Erickson Bobbie R. , Morgan Laura T. , Khristova Marina L. , Hartman Amy L. , Comer James A. , Rollin Pierre E. , Ksiazek Thomas G. , Nichol Stuart T.
Published: September 2008

Rift Valley fever (RVF) virus historically has caused widespread and extensive outbreaks of severe human and livestock disease throughout Africa, Madagascar, and the Arabian Peninsula. Following unusually heavy rainfall during the late autumn of 2006, reports of human and animal illness consistent with RVF virus infection emerged across semiarid regions of the Garissa District of northeastern Kenya and southern Somalia. Following initial RVF virus laboratory confirmation, a high-throughput RVF diagnostic facility was established at the Kenyan Central Veterinary Laboratories in Kabete, Kenya, to support the real-time identification of infected livestock and to facilitate outbreak response and control activities. A total of 3,250 specimens from a variety of animal species, including domesticated livestock (cattle, sheep, goats, and camels) and wildlife collected from a total of 55 of 71 Kenyan administrative districts, were tested by molecular and serologic assays. Evidence of RVF infection was found in 9.2% of animals tested and across 23 districts of Kenya, reflecting the large number of affected livestock and the geographic extent of the outbreak. The complete S, M, and/or L genome segment sequence was obtained from a total of 31 RVF virus specimens spanning the entire known outbreak period (December-May) and geographic areas affected by RVF virus activity. Extensive genomic analyses demonstrated the concurrent circulation of multiple virus lineages, gene segment reassortment, and the common ancestry of the 2006/2007 outbreak viruses with those from the 1997-1998 east African RVF outbreak. Evidence of recent increases in genomic diversity and effective population size 2 to 4 years prior to the 2006-2007 outbreak also was found, indicating ongoing RVF virus activity and evolution during the interepizootic/epidemic period. These findings have implications for further studies of basic RVF virus ecology and the design of future surveillance/diagnostic activities, and they highlight the critical need for safe and effective vaccines and antiviral compounds to combat this significant veterinary and public health threat.
Source mechanisms of vulcanian eruptions at Mt. Asama, Japan, inferred from volcano seismic signals
Authors: Ohminato Takao
Published: September 2008

During the 2004 Asama volcanic activity, five summit eruptions, accompanied by the broadband seismic signals, were observed. We re-analyse the broadband waveform data analysed by

Ohminato
et al.
(2006)

using relaxed restrictions. The results are essentially the same as those shown in the previous study. The results of the waveform inversions that assume a point source show that the force system is dominated by vertical single-force components. The source depths with dominant single-force components are 200–300 m beneath the summit crater. In the source-time history of the vertical single-force component, two downward forces separated by an upward force lasting for 5–6 s are clearly seen. We conduct a grid search for the best combination of two point sources, each consisting of a single-force component. The best waveform-match solution was obtained when one of them is positioned near the top of the conduit, and the other source is positioned 2000 m below the upper source. When a combination of single-force and moment-tensor components is assumed for the two-point source model, the moment source is located out of the vertical hypocentre distribution, suggesting a steeply inclined conduit.
The feasibility of generating low-frequency volcano seismicity by flow through a deformable channel
Authors: Rust A. C. , Balmforth N. J. , Mandre S.
Published: September 2008

Oscillations generated by flow of magmatic or hydrothermal fluids through tabular channels in elastic rocks are a possible source of low-frequency seismicity. We assess the conditions required to generate oscillations of approximately 1 Hz
via
hydrodynamic flow instabilities (roll waves), flow-destabilized standing waves set up on the elastic channel walls (wall modes), and unstable normal modes ringing in an adjacent fluid reservoir (clarinet modes). Stability criteria are based on physical and dimensional arguments, and discussion of destabilized elastic modes is supplemented with laboratory experiments of gas flow through a channel in a block of gelatine, and between a rigid plate and a rubber membrane. For each of the mechanisms considered, oscillations are generated if flow speeds exceed a critical value. Roll waves are waves of channel thickness variation that propagate in the direction of flow and are equivalent to traveling crack waves. The convective instability criterion is that the flow is faster than those travelling waves. Similarly, wall modes and clarinet modes require that the flow speed exceeds a critical value related to a wave speed (e.g. elastic or acoustic wave) multiplied by a geometrical factor. Flow destabilized modes offer a plausible explanation for low-frequency volcano seismicity, but there are limitations on what kind of standing waves comprises them.
Shallow-conduit dynamics at Stromboli Volcano, Italy, imaged from waveform inversions
Authors: Chouet Bernard , Dawson Phillip , Martini Marcello
Published: September 2008

Modelling of Very-Long-Period (VLP) seismic data recorded during explosive activity at Stromboli in 1997 provides an image of the uppermost 1 km of its volcanic plumbing system. Two distinct dyke-like conduit structures are identified, each representative of explosive eruptions from two different vents located near the northern and southern perimeters of the summit crater. Observed volumetric changes in the dykes are viewed as the result of a piston-like action of the magma associated with the disruption of a gas slug transiting through discontinuities in the dyke apertures. Accompanying these volumetric source components are single vertical forces resulting from an exchange of linear momentum between the source and the Earth. In the dyke system underlying the northern vent, a primary disruption site is observed at an elevation near 440 m where a bifurcation in the conduit occurs. At a depth of 80 m below sea level, a sharp corner in the conduit marks another location where the elastic response of the solid to the action of the upper source induces pressure and momentum changes in the magma. In the conduit underlying the southern vent, the junction of two inclined dykes with a sub-vertical dyke at 520 m elevation is a primary site of gas slug disruption, and another conduit corner 280 m below sea level represents a coupling location between the elastic response of the solid and fluid motion.
From sound waves to bubbling within a magma reservoir: comparison between eruptions at Etna (2001, Italy) and Kilauea (Hawaii)
Authors: Vergniolle S.
Published: September 2008

The 2001 eruption of Etna, prior to the flank eruption, has shown an alternation between episodes rich in gas, composed with a series of Strombolian explosions sometimes leading to a fire fountain, and repose periods. The regular alternation results from the coalescence of a foam trapped in the reservoir and periodically rebuilt prior to each episode. The degassing of a magma reservoir depends on bubble diameter, gas volume fraction, surface area and height of the reservoir. These four parameters are deduced from the measured gas flux, the timescale over which the gas flux decreases and the foam dynamics. The dimensionless foam thickness, 0.76 for a purely Strombolian episode, increases to 0.89 for an episode leading to a fire fountain, indicating a more efficient foam coalescence. At Etna, the bubble diameter, gas volume fraction, surface area and height of the reservoir are estimated at 0.50–0.59 mm, 0.25–0.39%, >0.20 km
2
and 97–220 m, respectively. At Kilauea, the excellent agreement between the prediction of the foam model, 0.94–1.2 km
2
and that resulting from deformations, 1 km
2
, reinforces the validity of the foam model qualitatively and quantitatively. The thickness of the degassing reservoir, 16–12 m in 1959, is now 1.3–1.1 km.
Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria
Authors: Baggaley Erin M. , Elliott Austin C. , Bruce Jason I. E.
Published: September 2008

Impairment of the normal spatiotemporal pattern of intracellular Ca2+ ([Ca2+]i) signaling, and in particular, the transition to an irreversible “Ca2+ overload” response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca2+ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H2O2) evokes a Ca2+ overload response and inhibition of plasma membrane Ca2+-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938–C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca2+]i clearance assay in which mitochondrial Ca2+ uptake was blocked with Ru-360, H2O2 (50 μM–1 mM) markedly inhibited the PMCA activity. This H2O2-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H2O2-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca2+ handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca2+ overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.
Effects of clenbuterol on contractility and Ca2+homeostasis of isolated rat ventricular myocytes
Authors: Siedlecka U. , Arora M. , Kolettis T. , Soppa G. K. R. , Lee J. , Stagg M. A. , Harding S. E. , Yacoub M. H. , Terracciano C. M. N.
Published: September 2008

Clenbuterol, a compound classified as a β2-adrenoceptor (AR) agonist, has been employed in combination with left ventricular assist devices (LVADs) to treat patients with severe heart failure. Previous studies have shown that chronic administration of clenbuterol affects cardiac excitation-contraction coupling. However, the acute effects of clenbuterol and the signaling pathway involved remain undefined. We investigated the acute effects of clenbuterol on isolated ventricular myocyte sarcomere shortening, Ca2+transients, and L-type Ca2+current and compared these effects to two other clinically used β2-AR agonists: fenoterol and salbutamol. Clenbuterol (30 μM) produced a negative inotropic response, whereas fenoterol showed a positive inotropic response. Salbutamol had no significant effects. Clenbuterol reduced Ca2+transient amplitude and L-type Ca2+current. Selective β1-AR blockade did not affect the action of clenbuterol on sarcomere shortening but significantly reduced contractility in the presence of fenoterol and salbutamol ( P < 0.05). Incubation with 2 μg/ml pertussis toxin significantly reduced the negative inotropic effects of 30 μM clenbuterol. In addition, overexpression of inhibitory G protein (Gi) by adenoviral transfection induced a stronger clenbuterol-mediated negative inotropic effect, suggesting the involvement of the Giprotein. We conclude that clenbuterol does not increase and, at high concentrations, significantly depresses contractility of isolated ventricular myocytes, an effect not seen with fenoterol or salbutamol. In its negative inotropism, clenbuterol predominantly acts through Gi, and the consequent downstream signaling pathways activation may explain the beneficial effects observed during chronic administration of clenbuterol in patients treated with LVADs.