bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+‐binding proteins tune Ca2+‐feedback to Cav1.3 channels in mouse auditory hair cells
Authors: Cui Guiying , Meyer Alexander C. , Calin‐Jageman Irina , Neef Jakob , Haeseleer Françoise , Moser Tobias , Lee Amy
Published: October 2007

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Cav1.3 voltage‐gated Ca2+ channels. Central to their role in this regard, Cav1.3 channels in inner hair cells show little Ca2+‐dependent inactivation, a fast negative feedback regulation by incoming Ca2+ ions, which depends on calmodulin association with the Ca2+ channel α1 subunit. Ca2+‐dependent inactivation characterizes nearly all voltage‐gated Ca2+ channels including Cav1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Cav1.3 in inner hair cells remains a mystery. Previously, we established calmodulin‐like Ca2+‐binding proteins in the brain and retina (CaBPs) as essential modulators of voltage‐gated Ca2+ channels. Here, we demonstrate that CaBPs differentially modify Ca2+ feedback to Cav1.3 channels in transfected cells and explore their significance for Cav1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca2+‐dependent inactivation of Cav1.3. CaBP1 and CaBP4 both interact with calmodulin‐binding sequences in Cav1.3, but CaBP4 more weakly inhibits Ca2+‐dependent inactivation than CaBP1. Ca2+‐dependent inactivation is marginally greater in inner hair cells from CaBP4−/− than from wild‐type mice, yet CaBP4−/− mice are not hearing‐impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild‐type and CaBP4−/− mice and therefore is positioned to modulate native Cav1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca2+ channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Cav1.3 Ca2+ currents required for auditory transmission.
Central and peripheral neuroimmune responses: hyporesponsiveness during pregnancy
Authors: Spencer Sarah J. , Mouihate Abdeslam , Galic Michael A. , Pittman Quentin J.
Published: October 2007

There are periods in the life of a healthy animal (including humans) when the febrile response to an immune challenge is suppressed. One such period is during late pregnancy, particularly around the time of parturition. In the 30 or so years since this ‘febrile hyporesponsiveness’ was first noted, much work has been done to investigate the mechanisms and adaptive significance of this phenomenon. In this review we present some insight into how and why the body deliberately re‐programmes itself to develop smaller fevers in response to an immune challenge and therefore to be potentially less successful at fighting infection.
Endogenous nitric oxide attenuates neutrally mediated cutaneous vasoconstriction
Authors: Shibasaki Manabu , Durand Sylvain , Davis Scott L. , Cui Jian , Low David A. , Keller David M. , Crandall Craig G.
Published: October 2007

Cutaneous vasoconstrictor responsiveness may be impaired by substance(s) directly or indirectly responsible for cutaneous active vasodilatation. In this study, we tested the hypothesis that endogenous nitric oxide (NO) attenuates the reduction in cutaneous vascular conductance (CVC) during an orthostatic challenge combined with whole‐body heating, as well as during whole‐body cooling. In protocol 1, healthy subjects were pretreated with an intradermal injection of botulinum toxin A (BTX) to block the release of neurotransmitters from nerves responsible for cutaneous active vasodilatation. On the experimental day, a microdialysis probe was placed at the BTX‐treated site as well as at two adjacent untreated sites. NG‐nitro‐l‐arginine methyl ester (l‐NAME, 10 mm) was perfused through the probe placed at the BTX‐treated site and at one untreated site. After confirmation of the absence of cutaneous vasodilatation at the BTX site during whole‐body heating, adenosine was infused through the microdialysis probe at this site to increase skin blood flow to a level similar to that at the untreated site. Subsequently, 30 and 40 mmHg lower‐body negative pressures (LBNPs) were applied. The reduction in CVC to LBNP was greatest at the BTX‐treated site (15.0 ± 2.4% of the maximum level (% max)), followed by the l‐NAME‐treated site (11.3 ± 2.6% max), and then the untreated site (3.8 ± 3.0% max; P < 0.05 for all comparisons). In protocol 2, two microdialysis membranes were inserted in the dermal space of one forearm. Adenosine alone was infused at one site while the other site received adenosine and l‐NAME. The reduction in CVC in response to whole‐body cooling was significantly greater at the l‐NAME‐treated site than at the adjacent adenosine alone site. These results suggest that endogenous NO is capable of attenuating cutaneous vasoconstrictor responsiveness.
Low glucose effects on rat carotid body chemoreceptor cells' secretory responses and action potential frequency in the carotid sinus nerve
Authors: Conde S. V. , Obeso A. , Gonzalez C.
Published: October 2007

Glucose deprivation (hypoglycaemia) is counterbalanced by a neuroendocrine response in order to induce fast delivery of glucose to blood. Some central neurons can sense glucose, but nevertheless the most important glucose sensors/glycaemia regulators are located outside the brain. Some recent experimental evidence obtained in carotid body (CB) slices and isolated chemoreceptor cells in culture supports a role for the CB in glucose sensing and presumably glucose homeostasis, but this role has been questioned on the basis of a lack of effect of low glucose on the carotid sinus nerve activity. This work was performed in an attempt to clarify if low glucose is or is not a stimulus for the rat CB chemoreceptors. Using freshly isolated intact CB preparations we have monitored the release of catecholamines (CAs) and ATP from chemoreceptor cells in response to several concentrations of glucose, as indices of chemoreceptor cell sensitivity to glycaemia, and the electrical activity in the carotid sinus nerve (CSN), as an index of reflex‐triggering output of the CB. We have observed that basal (20% O2) and hypoxia (7 and 10% O2)‐evoked release of CAs was identical in the presence of normal (5.55 mm) and low (3, 1 and 0 mm) glucose concentrations. 0 mm glucose did not activate the release of ATP from the CB, while hypoxia (5% O2) did. Basal and hypoxia (5% O2)‐induced CSN action potential frequency was identical with 5.55 and 1 mm glucose. Our results indicate that low glucose is not a direct stimulus for the rat carotid body chemoreceptors.
Adiponectin selectively inhibits oxytocin neurons of the paraventricular nucleus of the hypothalamus
Authors: Hoyda Ted D. , Fry Mark , Ahima Rexford S. , Ferguson Alastair V.
Published: October 2007

Adiponectin is an adipocyte derived hormone which acts in the brain to modulate energy homeostasis and autonomic function. The paraventricular nucleus of the hypothalamus (PVN) which plays a key role in controlling pituitary hormone secretion has been suggested to be a central target for adiponectin actions. A number of hormones produced by PVN neurons have been implicated in the regulation of energy homeostasis including oxytocin, corticotropin releasing hormone and thyrotropin releasing hormone. In the present study we investigated the role of adiponectin in controlling the excitability of magnocellular (MNC – oxytocin or vasopressin secreting) neurons within the PVN. Using RT‐PCR techniques we have shown expression of both adiponectin receptors in the PVN. Patch clamp recordings from MNC neurons in hypothalamic slices have also identified mixed (27% hyperpolarization, 42% depolarization) effects of adiponectin in modulating the excitability of the majority of MNC neurons tested. These effects are maintained when cells are placed in synaptic isolation using tetrodotoxin. Additionally we combined electrophysiological recordings with single cell RT‐PCR to examine the actions of adiponectin on MNC neurons which expressed oxytocin only, vasopressin only, or both oxytocin and vasopressin mRNA and assess the profile of receptor expression in these subgroups. Adiponectin was found to hyperpolarize 100% of oxytocin neurons tested (n= 6), while vasopressin cells, while all affected (n= 6), showed mixed responses. Further analysis indicates oxytocin neurons express both receptors (6/7) while vasopressin neurons express either both receptors (3/8) or one receptor (5/8). In contrast 6/6 oxytocin/vasopressin neurons were unaffected by adiponectin. Co‐expressing oxytocin and vasopressin neurons express neither receptor (4/6). The results presented in this study suggest that adiponectin plays specific roles in controlling the excitability oxytocin secreting neurons, actions which correlate with the current literature showing increased oxytocin secretion in the obese population.
Differential expression of Na+/K+‐ATPase α‐subunits in mouse hippocampal interneurones and pyramidal cells
Authors: Richards Kathryn S. , Bommert Kurt , Szabo Gabor , Miles Richard
Published: October 2007

The sodium pump (Na+/K+‐ATPase), maintains intracellular and extracellular concentrations of sodium and potassium by catalysing ATP. Three sodium pump α subunits, ATP1A1, ATP1A2 and ATP1A3, are expressed in brain. We compared their role in pyramidal cells and a subset of interneurones in the subiculum. Interneurones were identified by their expression of GFP under the GAD‐65 promoter. We used the sensitivity to the cardiac glycoside, ouabain, to discriminate between different α subunit isoforms. GFP‐positive interneurones were depolarized by nanomolar doses of ouabain, but higher concentrations were needed to depolarize pyramidal cells. Comparison of pump currents in these cells revealed a current sensitive to low doses of ouabain in interneurones, while micromolar doses of ouabain were needed to suppress the pump current in subicular pyramidal cells. As predicted, nanomolar doses of ouabain increased the frequency but not the amplitudes of IPSPs in pyramidal cells. Immunostaining confirmed a differential distribution of α‐subunits of the Na+/K+‐ATPase in subicular interneurones and pyramidal cells. In conclusion, these data suggest that while ATP1A3‐isoforms regulate sodium and potassium homeostasis in subicular interneurones, ATP1A1‐isoforms assume this function in pyramidal cells. This differential expression of sodium pump isoforms may contribute to differences in resting membrane potential of subicular interneurones and pyramidal cells.
Active properties of motoneurone dendrites: diffuse descending neuromodulation, focused local inhibition
Authors: Heckman C. J. , Hyngstrom Allison S. , Johnson Michael D.
Published: October 2007

The dendrites of spinal motoneurones are highly active, generating a strong persistent inward current (PIC) that has an enormous impact on processing of synaptic input. The PIC is subject to regulation by descending neuromodulatory systems releasing the monoamines serotonin and noradrenaline. At high monoaminergic drive levels, the PIC dominates synaptic integration, generating an intrinsic dendritic current that is as much as 5‐fold larger than the current entering via synapses. Without the PIC, motoneurone excitability is very low. Presumably, this descending control of the synaptic integration via the PIC is used to adjust the excitability (gain) of motoneurones for different motor tasks. A problem with this gain control is that monoaminergic input to the cord is very diffuse, affecting many motor pools simultaneously, probably including both agonists and antagonists. The PIC is, however, exquisitely sensitive to the reciprocal inhibition mediated by length sensitive muscle spindle Ia afferents and Ia interneurones. Reciprocal inhibition is tightly focused, shared only between strict mechanical antagonists, and thus can act to ‘sculpt’ specific movement patterns out of a background of diffuse neuromodulation. Thus it is likely that motoneurone gain is set by the interaction between diffuse descending neuromodulation and specific and focused local synaptic inhibitory circuits.
Ca2+‐dependent generation of mitochondrial reactive oxygen species serves as a signal for poly(ADP‐ribose) polymerase‐1 activation during glutamate excitotoxicity
Authors: Duan Yuntao , Gross Robert A. , Sheu Shey‐Shing
Published: October 2007

Mitochondrial Ca2+ uptake and poly(ADP‐ribose) polymerase‐1 (PARP‐1) activation are both required for glutamate‐induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca2+ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP‐1 activation in cultured striatal neurons. Based on the spatial profile of NMDA‐induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A2 or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca2+ from extracellular medium and blockage of mitochondrial Ca2+ uptake by either a mitochondrial uncoupler or a Ca2+ uniporter inhibitor. Furthermore, both DNA damage and PARP‐1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca2+ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca2+‐dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP‐1 activation, suggesting that concomitant mitochondrial Ca2+ uptake and PARP‐1 activation constitute a unified mechanism for excitotoxic neuronal death.
A β-Defensin Mutation Causes Black Coat Color in Domestic Dogs
Authors: Candille Sophie I. , Kaelin Christopher B. , Cattanach Bruce M. , Yu Bin , Thompson Darren A. , Nix Matthew A. , Kerns Julie A. , Schmutz Sheila M. , Millhauser Glenn L. , Barsh Gregory S.
Published: October 2007

Genetic analysis of mammalian color variation has provided fundamental insight into human biology and disease. In most vertebrates, two key genes,
Agouti
and
Melanocortin 1 receptor
(
Mc1r
), encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene is implicated, the
K
locus, whose genetic characteristics predict a previously unrecognized component of the melanocortin pathway. We identify the
K
locus as β-
defensin 103
(
CBD103
) and show that its protein product binds with high affinity to the Mc1r and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mice. These results expand the functional role of β-defensins, a protein family previously implicated in innate immunity, and identify an additional class of ligands for signaling through melanocortin receptors.
Survival of
Burkholderia pseudomallei
on Environmental Surfaces
Authors: Shams Alicia M. , Rose Laura J. , Hodges Lisa , Arduino Matthew J.
Published: October 2007

The survival of the biothreat agent
Burkholderia pseudomallei
on the surfaces of four materials was measured by culture and esterase activity analyses. The culture results demonstrated that this organism persisted for <24 h to <7 days depending on the material, bacterial isolate, and suspension medium. The persistence determined by analysis of esterase activity, as measured with a ScanRDI solid-phase cytometer, was always longer than the persistence determined by culture analysis.
Reconstitution of Glyphosate Resistance from a Split 5-Enolpyruvyl Shikimate-3-Phosphate Synthase Gene in
Escherichia coli
and Transgenic Tobacco
Authors: Dun Bao-Qing , Wang Xu-Jing , Lu Wei , Zhao Zhong-Lin , Hou Song-Na , Zhang Bao-Ming , Li Gui-Ying , Evans Thomas C. , Xu Ming-Qun , Lin Min
Published: October 2007

A highly
N
-phosphonomethylglycine (glyphosate)-resistant
Pseudomonas fluorescens
G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in
Escherichia coli
strain ER2799 and transgenic tobacco.
Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam
Authors: Van Thi Thu Hao , Moutafis George , Tran Linh Thuoc , Coloe Peter J.
Published: October 2007

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat samples and 18.0% of shellfish samples were contaminated with
Salmonella
spp., and more than 90% of all food sources contained
Escherichia coli
. The isolates were screened for antibiotic resistance against 15 antibiotics, and 50.5% of
Salmonella
isolates and 83.8% of
E. coli
isolates were resistant to at least one antibiotic. Isolates were examined for the presence of mobile genetic elements conferring antibiotic resistance. Fifty-seven percent of
E. coli
and 13% of
Salmonella
isolates were found to contain integrons, and some isolates contained two integrons. Sequencing results revealed that the integrons harbored various gene cassettes, including
aadA1
,
aadA2
, and
aadA5
(resistance to streptomycin and spectinomycin),
aacA4
(resistance to aminoglycosides), the dihydrofolate reductase gene cassettes
dhfrXII
,
dfrA1
, and
dhfrA17
(trimethoprim resistance), the beta-lactamase gene
bla
PSE1
(ampicillin resistance), and
catB3
(chloramphenicol resistance). Plasmids were also detected in all 23 antibiotic-resistant
Salmonella
isolates and in 33
E. coli
isolates. Thirty-five percent of the
Salmonella
isolates and 76% of the
E. coli
isolates contained plasmids of more than 95 kb, and some of the isolates contained two large plasmids. Conjugation experiments showed the successful transfer of all or part of the antibiotic resistance phenotypes among the
Salmonella
and
E. coli
food isolates. Our results show that enteric bacteria in raw food samples from Vietnam contain a pool of mobile genetic elements and that the transfer of antibiotic resistance can readily occur between similar bacteria.
Ni Uptake and Limitation in MarineSynechococcusStrains
Authors: Dupont Christopher L. , Barbeau Katherine , Palenik Brian
Published: October 2007

Ni accumulation and utilization were studied in two strains of marineSynechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO3−and can grow only slowly on NH4+without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marineSynechococcusand allProchlorococcusstrains is dependent upon Ni.
Association of
Vibrio cholerae
O1 El Tor and O139 Bengal with the Copepods
Acartia tonsa
and
Eurytemora affinis
Authors: Rawlings Tonya K. , Ruiz Gregory M. , Colwell Rita R.
Published: October 2007

The association of
Vibrio cholerae
with zooplankton has been suggested as an important factor in transmission of human epidemic cholera, and the ability to colonize zooplankton surfaces may play a role in the temporal variation and predominance of the two different serogroups (
V. cholerae
O1 El Tor and O139) in the aquatic environment. To date, interactions between specific serogroups and species of plankton remain poorly understood. Laboratory microcosm experiments were carried out to compare quantitatively the colonization of two copepod species,
Acartia tonsa
and
Eurytemora affinis
, by each of the epidemic serogroups.
V. cholerae
O1 consistently achieved higher abundances than
V. cholerae
O139 in colonizing adults of each copepod species as well as the multiple life stages of
E. affinis
. This difference in colonization may be significant in the general predominance of
V. cholerae
O1 in cholera epidemics in rural Bangladesh where water supplies are taken directly from the environment.
Multilocus Characterization Scheme for Shiga Toxin-Encoding Bacteriophages
Authors: Smith Darren L. , Wareing Brian M. , Fogg Paul C. M. , Riley Laura M. , Spencer Matthew , Cox Michael J. , Saunders Jon R. , McCarthy Alan J. , Allison Heather E.
Published: October 2007

Shiga toxin-producing
Escherichia coli
(STEC) strains are food-borne pathogens whose ability to produce Shiga toxin (Stx) is due to integration of Stx-encoding lambdoid bacteriophages. These Stx phages are both genetically and morphologically heterogeneous, and here we report the design and validation of a PCR-based multilocus typing scheme. PCR primer sets were designed for database variants of a range of key lambdoid bacteriophage genes and applied to control phages and 70
stx
+
phage preparations induced from a collection of STEC isolates. The genetic diversity residing within these populations could be described, and observations were made on the heterogeneity of individual gene targets, including the unexpected predominance of short-tailed phages with a highly conserved tail spike protein gene. Purified Stx phages can be profiled using this scheme, and the lambdoid phage-borne genes in induced STEC preparations can be identified as well as those residing in the noninducible prophage complement. The ultimate goal is to enable robust and realistically applicable epidemiological studies of Stx phages and their traits. The impact of Stx phage on STEC epidemiology is currently unknown.
Improved Succinic Acid Production in the Anaerobic Culture of an
Escherichia coli pflB ldhA
Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture
Authors: Wu Hui , Li Zhi-min , Zhou Li , Ye Qin
Published: October 2007

Escherichia coli
NZN111 is a
pflB ldhA
double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered
E. coli
strain.
CpxRA Regulates Mutualism and Pathogenesis in
Xenorhabdus nematophila
Authors: Herbert Erin E. , Cowles Kimberly N. , Goodrich-Blair Heidi
Published: October 2007

The CpxRA signal transduction system, which in
Escherichia coli
regulates surface structure assembly and envelope maintenance, is involved in the pathogenic and mutualistic interactions of the entomopathogenic bacterium
Xenorhabdus nematophila
. When Δ
cpxR1
cells were injected into
Manduca sexta
insects, the time required to kill 50% of the insects was twofold longer than the time observed for wild-type cells and the Δ
cpxR1
cells ultimately killed 16% fewer insects than wild-type cells killed. During mutualistic colonization of
Steinernema carpocapsae
nematodes, the Δ
cpxR1
mutant achieved colonization levels that were only 38% of the wild-type levels. Δ
cpxR1
cells exhibited an extended lag phase when they were grown in liquid LB or hemolymph, formed irregular colonies on solid medium, and had a filamentous cell morphology. A mutant with a
cpxRp-lacZ
fusion had peaks of expression in the log and stationary phases that were conversely influenced by CpxR; the Δ
cpxR1
mutant produced 130 and 17% of the wild-type β-galactosidase activity in the log and stationary phases, respectively. CpxR positively influences motility and secreted lipase activity, as well as transcription of genes necessary for mutualistic colonization of nematodes. CpxR negatively influences the production of secreted hemolysin, protease, and antibiotic activities, as well as the expression of
mrxA
, encoding the pilin subunit. Thus,
X. nematophila
CpxRA controls expression of envelope-localized and secreted products, and its activity is necessary for both mutualistic and pathogenic functions.
Survival of Indicator and Pathogenic Bacteria in Bovine Feces on Pasture
Authors: Sinton Lester W. , Braithwaite Robin R. , Hall Carollyn H. , Mackenzie Margaret L.
Published: October 2007

The survival of enteric bacteria was measured in bovine feces on pasture. In each season, 11 cow pats were prepared from a mixture of fresh dairy cattle feces and sampled for up to 150 days. Four pats were analyzed for
Escherichia coli
, fecal streptococci, and enterococci, and four inoculated pats were analyzed for
Campylobacter jejuni
and
Salmonella enterica
. Two pats were placed on drainage collectors, and another pat was fitted with a temperature probe. In the first 1 to 3 weeks, there were increases (up to 1.5 orders of magnitude) in the counts of enterococci (in four seasons),
E. coli
(three seasons), fecal streptococci (three seasons), and
S. enterica
(two seasons), but there was no increase in the counts of
C. jejuni
. Thereafter, the counts decreased, giving an average ranking of the times necessary for 90% inactivation of
C. jejuni
(6.2 days from deposition) < fecal streptococci (35 days) <
S. enterica
(38 days) <
E. coli
(48 days) < enterococci (56 days). The pat temperature probably influenced bacterial growth, but the pattern of increases and decreases was primarily determined by desiccation; growth occurred when the water content was greater than 80%, but at a water content of 70 to 75% counts decreased.
E. coli
and enterococcus regrowth appeared to result from pat rehydration. Of 20 monthly leaching losses of
E. coli
, 16 were <10% of the total counts in the pat, and 12 were <1%. Drainage losses of
C. jejuni
(generally <1%) were detected for only 1 to 2 months. Although enterococci exhibited the best survival rate, higher final counts suggested that
E. coli
is the more practical indicator of bovine fecal pollution.
Conserved Processes and Lineage-Specific Proteins in Fungal Cell Wall Evolution
Authors: Coronado Juan E. , Mneimneh Saad , Epstein Susan L. , Qiu Wei-Gang , Lipke Peter N.
Published: October 2007

The cell wall is a defining organelle that differentiates fungi from its sister clades in the opisthokont superkingdom. With a sensitive technique to align low-complexity protein sequences, we have identified 187 cell wall-related proteins in
Saccharomyces cerevisiae
and determined the presence or absence of homologs in 17 other fungal genomes. There were both conserved and lineage-specific cell wall proteins, and the degree of conservation was strongly correlated with protein function. Some functional classes were poorly conserved and lineage specific: adhesins, structural wall glycoprotein components, and unannotated open reading frames. These proteins are primarily those that are constituents of the walls themselves. On the other hand, glycosyl hydrolases and transferases, proteases, lipases, proteins in the glycosyl phosphatidyl-inositol-protein synthesis pathway, and chaperones were strongly conserved. Many of these proteins are also conserved in other eukaryotes and are associated with wall synthesis in plants. This gene conservation, along with known similarities in wall architecture, implies that the basic architecture of fungal walls is ancestral to the divergence of the ascomycetes and basidiomycetes. The contrasting lineage specificity of wall resident proteins implies diversification. Therefore, fungal cell walls consist of rapidly diversifying proteins that are assembled by the products of an ancestral and conserved set of genes.
Regulation of Ammonia Homeostasis by the Ammonium Transporter AmtA in
Dictyostelium discoideum
Authors: Yoshino Ryuji , Morio Takahiro , Yamada Yoko , Kuwayama Hidekazu , Sameshima Masazumi , Tanaka Yoshimasa , Sesaki Hiromi , Iijima Miho
Published: October 2007

Ammonia has been shown to function as a morphogen at multiple steps during the development of the cellular slime mold
Dictyostelium discoideum
; however, it is largely unknown how intracellular ammonia levels are controlled. In the
Dictyostelium
genome, there are five genes that encode putative ammonium transporters:
amtA
,
amtB
,
amtC
,
rhgA
, and
rhgB
. Here, we show that AmtA regulates ammonia homeostasis during growth and development. We found that cells lacking
amtA
had increased levels of ammonia/ammonium, whereas their extracellular ammonia/ammonium levels were highly decreased. These results suggest that AmtA mediates the excretion of ammonium. In support of a role for AmtA in ammonia homeostasis, AmtA mRNA is expressed throughout the life cycle, and its expression level increases during development. Importantly, AmtA-mediated ammonia homeostasis is critical for many developmental processes.
amtA
−
cells are more sensitive to NH
4
Cl than wild-type cells in inhibition of chemotaxis toward cyclic AMP and of formation of multicellular aggregates. Furthermore, even in the absence of exogenously added ammonia, we found that
amtA
−
cells produced many small fruiting bodies and that the viability and germination of
amtA
−
spores were dramatically compromised. Taken together, our data clearly demonstrate that AmtA regulates ammonia homeostasis and plays important roles in multiple developmental processes in
Dictyostelium
.
Schizosaccharomyces pombe
Sst4p, a Conserved Vps27/Hrs Homolog, Functions Downstream of Phosphatidylinositol 3-Kinase Pik3p To Mediate Proper Spore Formation
Authors: Onishi Masayuki , Iida Michihiro , Koga Takako , Yamada Sadayuki , Hirata Aiko , Iwaki Tomoko , Takegawa Kaoru , Fukui Yasuhisa , Tachikawa Hiroyuki
Published: October 2007

Sporulation of the fission yeast
Schizosaccharomyces pombe
is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated.
sst4
Δ asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of
Saccharomyces cerevisiae
Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation.
Spatial Differentiation in the Vegetative Mycelium of
Aspergillus niger
Authors: Levin Ana M. , de Vries Ronald P. , Conesa Ana , de Bekker Charissa , Talon Manuel , Menke Hildegard H. , van Peij Noel N. M. E. , Wösten Han A. B.
Published: October 2007

Fungal mycelia are exposed to heterogenic substrates. The substrate in the central part of the colony has been (partly) degraded, whereas it is still unexplored at the periphery of the mycelium. We here assessed whether substrate heterogeneity is a main determinant of spatial gene expression in colonies of
Aspergillus niger
. This question was addressed by analyzing whole-genome gene expression in five concentric zones of 7-day-old maltose- and xylose-grown colonies. Expression profiles at the periphery and the center were clearly different. More than 25% of the active genes showed twofold differences in expression between the inner and outermost zones of the colony. Moreover, 9% of the genes were expressed in only one of the five concentric zones, showing that a considerable part of the genome is active in a restricted part of the colony only. Statistical analysis of expression profiles of colonies that had either been or not been transferred to fresh xylose-containing medium showed that differential expression in a colony is due to the heterogeneity of the medium (e.g., genes involved in secretion, genes encoding proteases, and genes involved in xylose metabolism) as well as to medium-independent mechanisms (e.g., genes involved in nitrate metabolism and genes involved in cell wall synthesis and modification). Thus, we conclude that the mycelia of 7-day-old colonies of
A. niger
are highly differentiated. This conclusion is also indicated by the fact that distinct zones of the colony grow and secrete proteins, even after transfer to fresh medium.
Establishment of the Ambient pH Signaling Complex in
Aspergillus nidulans
: PalI Assists Plasma Membrane Localization of PalH
Authors: Calcagno-Pizarelli Ana M. , Negrete-Urtasun Susana , Denison Steven H. , Rudnicka Joanna D. , Bussink Henk-Jan , Múnera-Huertas Tatiana , Stanton Ljiljana , Hervás-Aguilar América , Espeso Eduardo A. , Tilburn Joan , Arst Herbert N. , Peñalva Miguel A.
Published: October 2007

The
Aspergillus nidulans
ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cys-containing motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null
palI32
mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both
Saccharomyces cerevisiae
and
A. nidulans
, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.
Architecture of the Sporulation-Specific
Cdc14
Promoter from the Oomycete
Phytophthora infestans
Authors: Ah-Fong Audrey M. V. , Xiang Qijun , Judelson Howard S.
Published: October 2007

The
Cdc14
gene of
Phytophthora infestans
is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development. To unravel the mechanisms regulating its transcription, mutated
Cdc14
promoters plus chimeras of selected
Cdc14
sequences and a minimal promoter were tested in stable transformants. This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression. All three repeats need to be present for activity, suggesting that they bind a transcription factor through a cooperative mechanism. Electrophoretic mobility shift assays indicated that the CTYAAC repeats are specifically bound by a protein in nuclear extracts. Evidence was also obtained for a second region within the promoter that activates
Cdc14
transcription during sporulation which does not involve those repeats. The CTYAAC motif also affects the specificity of transcription initiation. Wild-type
Cdc14
is transcribed from a major start site and minor site(s) located about 100 nt upstream of the major site. However, stepwise mutations through the CTYAAC triad caused a graded shift to the upstream sites, as did mutating bases surrounding the major start site; transcripts initiated from the upstream site remained sporulation specific. Replacing the
Cdc14
initiation region with the Inr-like region of the constitutive
Piexo1
gene had no apparent effect on the pattern of transcription. Therefore, this study reports the first motif determining sporulation-induced transcription in oomycetes and helps define oomycete core promoters.
Genetic Diversity of the Gene Cluster Encoding Longus, a Type IV Pilus of Enterotoxigenic
Escherichia coli
Authors: Gomez-Duarte Oscar G. , Chattopadhyay Sujay , Weissman Scott J. , Giron Jorge A. , Kaper James B. , Sokurenko Evgeni V.
Published: October 2007

Enterotoxigenic
Escherichia coli
(ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene
lngA
demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.
Introduction of the Foreign Transposon Tn
4560
in
Streptomyces coelicolor
Leads to Genetic Instability near the Native Insertion Sequence IS
1649
Authors: Widenbrant Elizabeth M. , Kao Camilla M.
Published: October 2007

We report an altered pattern of genetic instability for
Streptomyces coelicolor
when the bacterium harbored a foreign transposon, Tn
4560
. Deletions, amplifications, and circularizations of the linear 8.7-Mb chromosome occurred more frequently at sites adjacent to native insertion elements, notably IS
1649
. In contrast, deletions, amplifications, and circularizations of a wild-type strain happened at heterogeneous sites within the chromosome. In 50 strains examined, structural changes removed or duplicated hundreds of contiguous
S. coelicolor
genes, altering up to 33% of the chromosome.
S. coelicolor
shows a bias toward one type of genetic instability during this particular assault from the environment, the invasion of foreign DNA.
Genome Sequence of
Staphylococcus aureus
Strain Newman and Comparative Analysis of Staphylococcal Genomes: Polymorphism and Evolution of Two Major Pathogenicity Islands
Authors: Baba Tadashi , Bae Taeok , Schneewind Olaf , Takeuchi Fumihiko , Hiramatsu Keiichi
Published: October 2007

Strains of
Staphylococcus aureus
, an important human pathogen, display up to 20% variability in their genome sequence, and most sequence information is available for human clinical isolates that have not been subjected to genetic analysis of virulence attributes.
S. aureus
strain Newman, which was also isolated from a human infection, displays robust virulence properties in animal models of disease and has already been extensively analyzed for its molecular traits of staphylococcal pathogenesis. We report here the complete genome sequence of
S. aureus
Newman, which carries four integrated prophages, as well as two large pathogenicity islands. In agreement with the view that
S. aureus
Newman prophages contribute important properties to pathogenesis, fewer virulence factors are found outside of the prophages than for the highly virulent strain MW2. The absence of drug resistance genes reflects the general antibiotic-susceptible phenotype of
S. aureus
Newman. Phylogenetic analyses reveal clonal relationships between the staphylococcal strains Newman, COL, NCTC8325, and USA300 and a greater evolutionary distance to strains MRSA252, MW2, MSSA476, N315, Mu50, JH1, JH9, and RF122. However, polymorphism analysis of two large pathogenicity islands distributed among these strains shows that the two islands were acquired independently from the evolutionary pathway of the chromosomal backbones of staphylococcal genomes. Prophages and pathogenicity islands play central roles in
S. aureus
virulence and evolution.
Streptomyces coelicolor
Undergoes Spontaneous Chromosomal End Replacement
Authors: Widenbrant Elizabeth M. , Tsai Hsiu-Hui , Chen Carton W. , Kao Camilla M.
Published: October 2007

We report a previously unobserved form of genetic instability for
Streptomyces coelicolor
, the replacement of one chromosome end by the other end. These genetic changes occurred spontaneously in both a wild-type strain and strains harboring a foreign transposon. Deleted and duplicated DNA comprises up to 33% of the genome.
A Novel Peroxiredoxin Activity Is Located within the C-Terminal End of
Rhodospirillum rubrum
Adenylyltransferase
Authors: Jonsson Anders , Teixeira Pedro Filipe , Nordlund Stefan
Published: October 2007

Adenylyltransferase (GlnE) catalyzes the reversible adenylylation of glutamine synthetase. In this report we present, for the first time, evidence for a peroxiredoxin activity of
Rhodospirillum rubrum
GlnE, through the carboxyl-terminal AhpC/thiol-specific antioxidant (TSA) domain. The combination of GlnE and AhpC/TSA domains within the same polypeptide constitutes a unique domain architecture that has not previously been identified among proteobacteria.
New Target Genes Controlled by the
Bradyrhizobium japonicum
Two-Component Regulatory System RegSR
Authors: Lindemann Andrea , Moser Annina , Pessi Gabriella , Hauser Felix , Friberg Markus , Hennecke Hauke , Fischer Hans-Martin
Published: October 2007

RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In
Bradyrhizobium japonicum
, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene
nifA
, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and Δ
regR
cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in
B. japonicum
. Furthermore, transcription profiling of Δ
regR
bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.
Cysteine Metabolism-Related Genes and Bacterial Resistance to Potassium Tellurite
Authors: Fuentes Derie E. , Fuentes Eugenia L. , Castro Miguel E. , Pérez José M. , Araya Manuel A. , Chasteen Thomas G. , Pichuantes Sergio E. , Vásquez Claudio C.
Published: October 2007

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of
Geobacillus stearothermophilus
V cysteine-related genes
cobA, cysK
, and
iscS
and
Escherichia coli
cysteine regulon genes under conditions that included the addition of K
2
TeO
3
to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.
Cooperative Transport between NukFEG and NukH in Immunity against the Lantibiotic Nukacin ISK-1 Produced by
Staphylococcus warneri
ISK-1
Authors: Okuda Ken-ichi , Aso Yuji , Nakayama Jiro , Sonomoto Kenji
Published: October 2007

Nukacin ISK-1 is a lantibiotic produced by
Staphylococcus warneri
ISK-1. Previous studies have reported that the self-protection system of the nukacin ISK-1 producer involves the cooperative function of the ABC transporter NukFEG and the lantibiotic-binding immunity protein NukH. In this study, the cooperative mechanism between NukFEG and NukH was characterized by using fluorescein-4-isothiocyanate (FITC)-labeled nukacin ISK-1 (FITC-nuk) to clarify the localization of nukacin ISK-1 in the immunity process.
Lactococcus lactis
recombinants expressing
nukFEGH
,
nukFEG
, or
nukH
showed immunity against FITC-nuk, suggesting that FITC-nuk was recognized by the self-protection system against nukacin ISK-1. Analysis of the interaction between FITC-nuk and energy-deprived cells of the
L. lactis
recombinants showed that FITC-nuk specifically bound to cells expressing
nukH
. The interaction between FITC-nuk and
nukH
-expressing cells was inhibited by the addition of unlabeled nukacin ISK-1 and its derivatives with deletions of the N-terminal tail region, but not by the addition of a synthesized N-terminal tail region. This suggests that the NukH protein recognizes the C-terminal ring region of nukacin ISK-1. The addition of glucose to
nukFEGH
-expressing cells treated with FITC-nuk resulted in a time-dependent decrease in fluorescence intensity, indicating that FITC-nuk was transported from the cell membrane by the NukFEG protein. These results revealed that after being captured by NukH in an energy-independent manner, nukacin ISK-1 was transported to the extracellular space by NukFEG in an energy-dependent manner.