bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Exercise in Preventing and Treating Gestational Diabetes: A Comprehensive Review and Recommendations for Future Research
Authors: DiNallo Jennifer M. , Downs Danielle Symons
Published: March 2008

The purposes of this study were to extensively review the gestational diabetes mellitus (GDM) and exercise literature and to provide recommendations for future research. We found (1) that exercise improved glucose utilization, decreased the need for insulin, or increased cardiorespiratory fitness in 62% of the intervention/treatment studies; (2) that pre‐pregnancy exercise was associated with GDM risk reduction in 45% of the correlational studies; (3) a greater risk reduction for overweight women in 27% of the correlational studies; and (4) a lack of consistency in reporting study characteristics and outcomes across all study types. Findings illustrate the positive effect of exercise on GDM outcomes, and the need for more consistency in data reporting across studies to systematically determine the protective mechanism and causal pathway of exercise for preventing GDM.
A sympathetic view of the sympathetic nervous system and human blood pressure regulation
Authors: Joyner Michael J. , Charkoudian Nisha , Wallin B. Gunnar
Published: March 2008

New ideas about the relative importance of the autonomic nervous system (and especially its sympathetic arm) in long‐term blood pressure regulation are emerging. It is well known that mean arterial blood pressure is normally regulated in a fairly narrow range at rest and that blood pressure is also able to rise and fall ‘appropriately’ to meet the demands of various forms of mental, emotional and physical stress. By contrast, blood pressure varies widely when the autonomic nervous system is absent or when key mechanisms that govern it are destroyed. However, 24 h mean arterial pressure is still surprisingly normal under these conditions. Thus, the dominant idea has been that the kidney is the main long‐term regulator of blood pressure and the autonomic nervous system is important in short‐term regulation. However, this ‘renocentric’ scheme can be challenged by observations in humans showing that there is a high degree of individual variability in elements of the autonomic nervous system. Along these lines, the level of sympathetic outflow, the adrenergic responsiveness of blood vessels and individual haemodynamic patterns appear to exist in a complex, but appropriate, balance in normotension. Furthermore, evidence from animals and humans has now clearly shown that the sympathetic nervous system can play an important role in longer term blood pressure regulation in both normotension and hypertension. Finally, humans with high baseline sympathetic traffic might be at increased risk for hypertension if the ‘balance’ among factors deteriorates or is lost. In this context, the goal of this review is to encourage a comprehensive rethinking of the complexities related to long‐term blood pressure regulation in humans and promote finer appreciation of physiological relationships among the autonomic nervous system, vascular function, ageing, metabolism and blood pressure.
Altered central catecholaminergic transmission and cardiovascular disease
Authors: Kasparov S. , Teschemacher A. G.
Published: March 2008

Numerous studies, some of which date back more than three decades, have established a link between disorders of the cardiovascular system and the catecholaminergic system of the brain. Central noradrenergic (and putative adrenergic) neurones are involved in numerous brain functions, and there appears to be more than one mechanism via which a dysfunction of central nor/adrenergic signalling may be detrimental to the cardiovascular system. Moreover, in some cases, such as essential hypertension, altered noradrenergic transmission could play a causative role. Numerous controversies are evident throughout the literature, which are very difficult to explain without much better understanding of the basic physiology of central noradrenergic transmission. Recently, using a combination of novel molecular, electrochemical and imaging techniques, we have started to unravel how noradrenergic neurones in the brain store and release their transmitter. Targeted long‐term modulation of specific noradrenergic cell groups in defined brain areas using viral gene transfer is helping to clarify the links between central catecholamines and cardiovascular control in health and disease. These studies may reveal new therapeutic strategies for various cardiovascular diseases which are accompanied by heightened sympathetic nerve activity.
A trip to the end of the universe and the twin “paradox”
Authors: Müller Thomas , King Andreas , Adis Daria
Published: March 2008

Special relativity offers the possibility of going on a trip to the center of our galaxy or even to the end of our universe within a lifetime. On the basis of the well known twin paradox, we discuss uniformly accelerated motion and emphasize the local perspective of each twin concerning the interchange of light signals between both twins as well as their different views of the stellar sky. For this purpose we developed two Java applets that students can use to explore interactively and understand the topics presented here.
Experiences using the open-source learning content management and assessment system LON-CAPA in introductory physics courses
Authors: Kortemeyer Gerd , Kashy Edwin , Benenson Walter , Bauer Wolfgang
Published: March 2008

We discuss the development and functionality of the LON-CAPA system with a particular focus on its homework and examination functionality. We also describe its more general approach to course management and its infrastructure for course content sharing and reuse. We then focus on measures of student learning and the effectiveness of different content types.
Functional
nodFE
Genes Are Present in
Sinorhizobium
sp. Strain MUS10, a Symbiont of the Tropical Legume
Sesbania rostrata
Authors: Krishnan Hari B. , Chronis Demosthenis
Published: March 2008

We have cloned the
nodFE
operon from
Sinorhizobium
sp. strain MUS10. MUS10 NodF shows sequence homology to acyl carrier protein and enables an
S. meliloti nodF
mutant to effectively nodulate alfalfa. Our results demonstrate the occurrence of
nodFE
in a symbiont that nodulates a legume host not belonging to the galegoid group.
Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the
Bacillus thuringiensis
Insecticidal Toxin Cry1Aa
Authors: Girard Frédéric , Vachon Vincent , Préfontaine Gabrielle , Marceau Lucie , Su Yanhui , Larouche Geneviève , Vincent Charles , Schwartz Jean-Louis , Masson Luke , Laprade Raynald
Published: March 2008

Helix α4 of
Bacillus thuringiensis
Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate
Manduca sexta
larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from
M
.
sexta
. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.
Comparison of BGM and PLC/PRC/5 Cell Lines for Total Culturable Viral Assay of Treated Sewage
Authors: Rodríguez Roberto A. , Gundy Patricia M. , Gerba Charles P.
Published: March 2008

The objective of this study was to compare PLC/PRF/5 and BGM cell lines for use in a total culturable viral assay (TCVA) of treated sewage effluents. Samples were collected before and after chlorination from an activated sludge wastewater treatment plant and from the effluent of a high-rate enhanced flocculation system, followed by UV light disinfection. Cell monolayers were observed for cytopathic effect (CPE) after two passages of 14 days each. Monolayers exhibiting viral CPE were tested for the presence of adenoviruses and enteroviruses by PCR or reverse transcription-PCR. Eight percent of the samples exhibited CPE on BGM cells, and 57% showed CPE on PLC/PRF/5 cells. Only enteroviruses were detected on the BGM cells, while 30% and 52% of the samples were positive for enteroviruses and adenoviruses, respectively, on the PLC/PRF/5 cells. Thirty percent of the samples were positive for both adenoviruses and enteroviruses in chlorinated activated sludge effluent. Thirty percent of the samples were positive for adenoviruses in the UV treatment effluent, but no enteroviruses were detected. In conclusion, the PLC/PRF/5 cells were more susceptible than BGM cells to viruses found in treated sewage. The use of BGM cells for TCVA may underestimate viral concentration in sewage effluent samples. The PLC/PRF/5 cells were more susceptible to adenoviruses, which is important in the evaluation of UV disinfection systems because adenoviruses are highly resistant to UV inactivation.
Feasibility of Transferring Fluorescent In Situ Hybridization Probes to an 18S rRNA Gene Phylochip and Mapping of Signal Intensities
Authors: Metfies Katja , Medlin Linda K.
Published: March 2008

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using
Prymenisum parvum
as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.
Cultivation and Ecosystem Role of a Marine
Roseobacter
Clade-Affiliated Cluster Bacterium
Authors: Mayali Xavier , Franks Peter J. S. , Azam Farooq
Published: March 2008

Isolation and cultivation are a crucial step in elucidating the physiology, biogeochemistry, and ecosystem role of microorganisms. Many abundant marine bacteria, including the widespread
Roseobacter
clade-affiliated (RCA) cluster group, have not been cultured with traditional methods. Using novel techniques of cocultivation with algal cultures, we have accomplished successful isolation and propagation of a strain of the RCA cluster. Our experiments revealed that, in addition to growing on alga-excreted organic matter, additions of washed bacterial cells led to significant biomass decrease of dinoflagellate cultures as measured by in vivo fluorescence. Bacterial filtrate did not adversely affect the algal cultures, suggesting attachment-mediated activity. Using an RCA cluster-specific rRNA probe, we documented increasing attachment of these algicidal bacteria during a dinoflagellate bloom, with a maximum of 70% of the algal cells colonized just prior to bloom termination. Cross-correlation analyses between algal abundances and RCA bacterial colonization were statistically significant, in agreement with predator-prey models suggesting that RCA cluster bacteria caused algal bloom decline. Further investigation of molecular databases revealed that RCA cluster bacteria were numerically abundant during algal blooms sampled worldwide. Our findings suggest that the widespread RCA cluster bacteria may exert significant control over phytoplankton biomass and community structure in the oceans. We also suggest that coculture with phytoplankton may be a useful strategy to isolate and successfully grow previously uncultured but ecologically abundant marine heterotrophs.
Quantifying Genes and Transcripts To Assess the In Situ Physiology of “
Dehalococcoides
” spp. in a Trichloroethene-Contaminated Groundwater Site
Authors: Lee Patrick K. H. , Macbeth Tamzen W. , Sorenson Kent S. , Deeb Rula A. , Alvarez-Cohen Lisa
Published: March 2008

Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (
tceA
,
vcrA
, and
bvcA
) as biomarkers of “
Dehalococcoides
” spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation.
Dehalococcoides
cells carrying the
tceA
,
vcrA
, and
bvcA
genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major
Dehalococcoides
metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total
Dehalococcoides
population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among
Dehalococcoides
cells. The
Dehalococcoides
cells containing the
tceA
gene consistently lagged behind other
Dehalococcoides
cells in population numbers and made up less than 5% of the total
Dehalococcoides
population, whereas the
vcrA
- and
bvcA
-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the
bvcA
and
vcrA
genes were consistently highly expressed in all samples examined, while the
tceA
transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the
bvcA
- and
vcrA
-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (
FtL-RDase-1638
) identified in the cDNA clone library tracked closely in field samples with abundance of the
bvcA
gene, suggesting that the
FtL-RDase-1638
gene was likely colocated in genomes containing the
bvcA
gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of
Dehalococcoides
strains and their associated activity.
Complete Genome Sequence of
Nitrobacter hamburgensis
X14 and Comparative Genomic Analysis of Species within the Genus
Nitrobacter
Authors: Starkenburg Shawn R. , Larimer Frank W. , Stein Lisa Y. , Klotz Martin G. , Chain Patrick S. G. , Sayavedra-Soto Luis A. , Poret-Peterson Amisha T. , Gentry Mira E. , Arp Daniel J. , Ward Bess , Bottomley Peter J.
Published: March 2008

The alphaproteobacterium
Nitrobacter hamburgensis
X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the
Nitrobacter hamburgensis
X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between
N. hamburgensis
and the finished and draft genome sequences of
Nitrobacter winogradskyi
and
Nitrobacter
sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to
N. hamburgensis
and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet ∼21 kb of a ∼28-kb “autotrophic” island on the largest plasmid was conserved in the chromosomes of
Nitrobacter winogradskyi
Nb-255 and
Nitrobacter
sp. strain Nb-311A. The
N. hamburgensis
chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome
b
561
, and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A
Nitrobacter
“subcore” genome was also constructed by removing homologs found in strains of the closest evolutionary relatives,
Bradyrhizobium japonicum
and
Rhodopseudomonas palustris
. Among the
Nitrobacter
subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in
Nitrobacter
.
Biochemical and Molecular Characterization of a Novel Type of Mutanase from
Paenibacillus
sp. Strain RM1: Identification of Its Mutan-Binding Domain, Essential for Degradation of
Streptococcus mutans
Biofilms
Authors: Shimotsuura Isao , Kigawa Hiromitsu , Ohdera Motoyasu , Kuramitsu Howard K. , Nakashima Syozi
Published: March 2008

A novel type of mutanase (termed mutanase RM1) was isolated from
Paenibacillus
sp. strain RM1. The purified enzyme specifically hydrolyzed α-1,3-glucan (mutan) and effectively degraded biofilms formed by
Streptococcus mutans
, a major etiologic agent in the progression of dental caries, even following brief incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in
Escherichia coli
demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity whereas the C-terminal domain was responsible for mutanase activity but had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.
New Triplex Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Feces
Authors: Schönenbrücher H. , Abdulmawjood A. , Failing K. , Bülte M.
Published: March 2008

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2sequences ofMycobacterium aviumsubsp.paratuberculosisfrom bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105M. aviumsubsp.paratuberculosisstrains of bovine, ovine, and human origin and 100 non-M. aviumsubsp.paratuberculosisstrains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogramM. aviumsubsp.paratuberculosisDNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection ofM. aviumsubsp.paratuberculosisDNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgband locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection ofM. aviumsubsp.paratuberculosisfrom bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknownM. aviumsubsp.paratuberculosisstatus resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.
Molecular Epidemiology of
Bacillus anthracis
: Determining the Correct Origin
Authors: Pilo Paola , Perreten Vincent , Frey Joachim
Published: March 2008

We analyzed and compared strains of
Bacillus anthracis
isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human
B. anthracis
strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of
B. anthracis
strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.
Global Response to Desiccation Stress in the Soil Actinomycete
Rhodococcus jostii
RHA1
Authors: LeBlanc Justin C. , Gonçalves Edmilson R. , Mohn William W.
Published: March 2008

Rhodococcus jostii
RHA1 is a soil-residing actinomycete with many favorable metabolic capabilities that make it an ideal candidate for the bioremediation of contaminated soils. Arguably the most basic requirement for life is water, yet some nonsporulating bacteria, like RHA1, can survive lengthy droughts. Here we report the first transcriptomic analysis of a gram-positive bacterium during desiccation. Filtered RHA1 cells incubated at either low relative humidity (20%), as an air-drying treatment, or high relative humidity (100%), as a control, were transcriptionally profiled over a comprehensive time series. Also, the morphology of RHA1 cells was characterized by cryofixation scanning electron microscopy during each treatment. Desiccation resulted in a transcriptional response of approximately 8 times more differentially regulated genes than in the control (819 versus 106 genes, respectively). Genes that were differentially expressed during only the desiccation treatment primarily had expression profiles that were maximally up-regulated upon complete drying of the cells. The microarray expression ratios for some of the highly up-regulated genes were verified by reverse transcriptase quantitative PCR. These genes included
dps1
, encoding an oxidative stress protection protein which has not previously been directly associated with desiccation, and the two genes encoding sigma factors SigF1 and SigF3, possibly involved in the regulatory response to desiccation. RHA1 cells also induced the biosynthetic pathway for the compatible solute ectoine. These desiccation-specific responses represent the best candidates for important mechanisms of desiccation resistance in RHA1.
Analysis of Neurotoxin Cluster Genes in
Clostridium botulinum
Strains Producing Botulinum Neurotoxin Serotype A Subtypes
Authors: Jacobson Mark J. , Lin Guangyun , Raphael Brian , Andreadis Joanne , Johnson Eric A.
Published: March 2008

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of
Clostridium botulinum
encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA
−
Orfx
+
A1). These sequences were compared to the known neurotoxin cluster sequences of
C. botulinum
strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA
−
Orfx
+
A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the
ntnh
gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the
ntnh
genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the
ntnh
gene was assigned.
Highly Selective and Rapid Arsenic Removal by Metabolically Engineered
Escherichia coli
Cells Expressing
Fucus vesiculosus
Metallothionein
Authors: Singh Shailendra , Mulchandani Ashok , Chen Wilfred
Published: March 2008

An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga
Fucus vesiculosus
was expressed in
Escherichia coli
, resulting in 30- and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting
E. coli
cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA.
Role of PUG1 in Inducible Porphyrin and Heme Transport in
Saccharomyces cerevisiae
Authors: Protchenko Olga , Shakoury-Elizeh Minoo , Keane Patricia , Storey Joshua , Androphy Rachel , Philpott Caroline C.
Published: March 2008

Unlike pathogenic fungi, the budding yeast
Saccharomyces cerevisiae
is not efficient at using heme as a nutritional source of iron. Here we report that for this yeast, heme uptake is induced under conditions of heme starvation. Heme synthesis requires oxygen, and yeast grown anaerobically exhibited an increased uptake of hemin. Similarly, a strain lacking aminolevulinate synthase exhibited a sixfold increase in hemin uptake when grown without 2-aminolevulinic acid. We used microarray analysis of cells grown under reduced oxygen tension or reduced intracellular heme conditions to identify candidate genes involved in heme uptake. Surprisingly, overexpression of
PUG1
(protoporphyrin uptake gene 1) resulted in reduced utilization of exogenous heme by a heme-deficient strain and, conversely, increased the utilization of protoporphyrin IX. Pug1p was localized to the plasma membrane by indirect immunofluorescence and subcellular fractionation. Strains overexpressing
PUG1
exhibited decreased accumulation of [
55
Fe]hemin but increased accumulation of protoporphyrin IX compared to the wild-type strain. To measure the effect of
PUG1
overexpression on intracellular heme pools, we used a
CYC1
-
lacZ
reporter, which is activated in the presence of heme, and we monitored the activity of a heme-containing metalloreductase, Fre1p, expressed from a constitutive promoter. The data from these experiments were consistent with a role for Pug1p in inducible protoporphyrin IX influx and heme efflux.
DictyosteliumAurora Kinase Has Properties of both Aurora A and Aurora B Kinases
Authors: Li Hui , Chen Qian , Kaller Markus , Nellen Wolfgang , Gräf Ralph , De Lozanne Arturo
Published: March 2008

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show thatDictyostelium discoideumcontains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.
RcoM: A New Single-Component Transcriptional Regulator of CO Metabolism in Bacteria
Authors: Kerby Robert L. , Youn Hwan , Roberts Gary P.
Published: March 2008

Genomic analysis suggested the existence of a CO-sensing bacterial transcriptional regulator that couples an N-terminal PAS fold domain to a C-terminal DNA-binding LytTR domain. UV/visible-light spectral analyses of heterologously expressed, purified full-length proteins indicated that they contained a hexacoordinated
b
-type heme moiety that avidly binds CO and NO. Studies of protein variants strongly suggested that the PAS domain residues His74 and Met104 serve as the heme Fe(II) axial ligands, with displacement of Met104 upon binding of the gaseous effectors. Two RcoM (
r
egulator of
CO
m
etabolism) homologs were shown to function in vivo as CO sensors capable of regulating an aerobic CO oxidation (
cox
) regulon. The genetic linkage of
rcoM
with both aerobic (
cox
) and anaerobic (
coo
) CO oxidation systems suggests that in different organisms RcoM proteins may control either regulon type.
The Legacy of HfrH: Mutations in the Two-Component System CreBC Are Responsible for the Unusual Phenotype of an
Escherichia coli arcA
Mutant
Authors: Nikel Pablo I. , de Almeida Alejandra , Pettinari M. Julia , Méndez Beatriz S.
Published: March 2008

Strains derived from HfrH carrying the
arcA2
null mutation exhibit a higher respiratory rate, enhanced glucose consumption, and a more-reduced intracellular redox state than
arcA
deletion mutants of a different lineage. The phenotype of the
arcA2
mutants was due to the presence of a
creC
constitutive mutation introduced by P1 transduction.
RhlA Converts β-Hydroxyacyl-Acyl Carrier Protein Intermediates in Fatty Acid Synthesis to the β-Hydroxydecanoyl-β-Hydroxydecanoate Component of Rhamnolipids inPseudomonas aeruginosa
Authors: Zhu Kun , Rock Charles O.
Published: March 2008

Pseudomonas aeruginosasecretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of β-hydroxydecanoyl-β-hydroxydecanoate from two molecules of β-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily β-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity inP. aeruginosaincreases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for β-hydroxydecanoyl-ACP. InEscherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.
ORF334 in
Vibrio
Phage KVP40 Plays the Role of gp27 in T4 Phage To Form a Heterohexameric Complex
Authors: Nemoto Mai , Mio Kazuhiro , Kanamaru Shuji , Arisaka Fumio
Published: March 2008

KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate.
Glutamate Metabolism inBacillus subtilis: Gene Expression and Enzyme Activities Evolved To Avoid Futile Cycles and To Allow Rapid Responses to Perturbations of the System
Authors: Commichau Fabian M. , Gunka Katrin , Landmann Jens J. , Stülke Jörg
Published: March 2008

Glutamate is a central metabolite in all organisms since it provides the link between carbon and nitrogen metabolism. InBacillus subtilis, glutamate is synthesized exclusively by the glutamate synthase, and it can be degraded by the glutamate dehydrogenase. InB. subtilis, the major glutamate dehydrogenase RocG is expressed only in the presence of arginine, and the bacteria are unable to utilize glutamate as the only carbon source. In addition torocG, a second cryptic gene (gudB) encodes an inactive glutamate dehydrogenase. Mutations inrocGresult in the rapid accumulation ofgudB1suppressor mutations that code for an active enzyme. In this work, we analyzed the physiological significance of this constellation of genes and enzymes involved in glutamate metabolism. We found that the weak expression ofrocGin the absence of the inducer arginine is limiting for glutamate utilization. Moreover, we addressed the potential ability of the active glutamate dehydrogenases ofB. subtilisto synthesize glutamate. Both RocG and GudB1 were unable to catalyze the anabolic reaction, most probably because of their very highKmvalues for ammonium. In contrast, theEscherichia coliglutamate dehydrogenase is able to produce glutamate even in the background of aB. subtiliscell.B. subtilisresponds to any mutation that interferes with glutamate metabolism with the rapid accumulation of extragenic or intragenic suppressor mutations, bringing the glutamate supply into balance. Similarly, with the presence of a cryptic gene, the system can flexibly respond to changes in the external glutamate supply by the selection of mutations.
Genomic and Functional Analysis of ICE
Pda
Spa1, a Fish-Pathogen-Derived SXT-Related Integrating Conjugative Element That Can Mobilize a Virulence Plasmid
Authors: Osorio Carlos R. , Marrero Joeli , Wozniak Rachel A. F. , Lemos Manuel L. , Burrus Vincent , Waldor Matthew K.
Published: March 2008

Integrating conjugative elements (ICEs) are self-transmissible mobile elements that transfer between bacteria via conjugation and integrate into the host chromosome. SXT and related ICEs became prevalent in Asian
Vibrio cholerae
populations in the 1990s and play an important role in the dissemination of antibiotic resistance genes in
V. cholerae
. Here, we carried out genomic and functional analyses of ICE
Pda
Spa1, an SXT-related ICE derived from a Spanish isolate of
Photobacterium damselae
subsp.
piscicida
, the causative agent of fish pasteurellosis. The ∼102-kb DNA sequence of ICE
Pda
Spa1 shows nearly 97% DNA sequence identity to SXT in genes that encode essential ICE functions, including integration and excision, conjugal transfer, and regulation. However, ∼25 kb of ICE
Pda
Spa1 DNA, including a tetracycline resistance locus, is not present in SXT. Most ICE
Pda
Spa1-specific DNA is inserted at loci where other SXT-related ICEs harbor element-specific DNA. ICE
Pda
Spa1 excises itself from the chromosome and is transmissible to other
Photobacterium
strains, as well as to
Escherichia coli
, in which it integrates into
prfC
. Interestingly, the
P. damselae
virulence plasmid pPHDP10 could be mobilized from
E. coli
in an ICE
Pda
Spa1-dependent fashion via the formation of a cointegrate between pPHDP10 and ICE
Pda
Spa1. pPHDP10-Cm integrated into ICE
Pda
Spa1 in a non-site-specific fashion independently of RecA. The ICE
Pda
Spa1::pPHDP10 cointegrates were stable, and markers from both elements became transmissible at frequencies similar to those observed for the transfer of ICE
Pda
Spa1 alone. Our findings reveal the plasticity of ICE genomes and demonstrate that ICEs can enable virulence gene transfer.
Topology and Function of CcmD in Cytochrome
c
Maturation
Authors: Richard-Fogal Cynthia L. , Frawley Elaine R. , Kranz Robert G.
Published: March 2008

The system I cytochrome
c
biogenesis pathway requires CcmD, a small polypeptide of 69 residues in
Escherichia coli
. Here it is shown that CcmD is a component of the CcmABC ATP-binding cassette transporter complex. CcmD is not necessary for the CcmC-dependent transfer of heme to CcmE in the periplasm or for interaction of CcmE with CcmABC. CcmD is absolutely required for the release of holo-CcmE from the CcmABCD complex. Evidence is presented that the topology of CcmD in the cytoplasmic membrane is the N terminus outside and the C terminus inside with one transmembrane domain.
Roles of Charged Residues in the C-Terminal Region of PomA, a Stator Component of the Na
+
-Driven Flagellar Motor
Authors: Obara Madoka , Yakushi Toshiharu , Kojima Seiji , Homma Michio
Published: March 2008

Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in
Escherichia coli
. Mutational studies have indicated that the Na
+
-driven motor of
Vibrio alginolyticus
may incorporate interactions similar to those of the
E. coli
motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na
+
-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na
+
-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of
V. alginolyticus
or with the Na
+
-driven chimeric motor of
E. coli
. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type
V. alginolyticus
. The R207E mutant conferred motility with the motor of
V. alginolyticus
but not with the chimeric motor of
E. coli
. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in
V. alginolyticus
but was greatly reduced in
E. coli
. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex.
Structural Analysis and Biosynthesis Gene Cluster of an Antigenic Glycopeptidolipid fromMycobacterium intracellulare
Authors: Fujiwara Nagatoshi , Nakata Noboru , Naka Takashi , Yano Ikuya , Doe Matsumi , Chatterjee Delphi , McNeil Michael , Brennan Patrick J. , Kobayashi Kazuo , Makino Masahiko , Matsumoto Sohkichi , Ogura Hisashi , Maeda Shinji
Published: March 2008

Mycobacterium avium-Mycobacterium intracellularecomplex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at theallo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived fromM. intracellularewas determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novelN-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2′-methyl-3′-hydroxy-4′-methoxy-pentanoyl-amido-3,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→2)-6-deoxy-α-l-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. AnM. aviumserotype 1 strain was transformed with cosmid clone no. 253 containinggtfB-drrCofM. intracellulareserotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.
A New Lineage of Halophilic, Wall-Less, Contractile Bacteria from a Brine-Filled Deep of the Red Sea
Authors: Antunes André , Rainey Fred A. , Wanner Gerhard , Taborda Marco , Pätzold Jürgen , Nobre M. Fernanda , da Costa Milton S. , Huber Robert
Published: March 2008

A novel strictly anaerobic bacterium designated strain SSD-17B
T
was isolated from the hypersaline brine-sediment interface of the Shaban Deep, Red Sea. Cells were pleomorphic but usually consisted of a central coccoid body with one or two “tentacle-like” protrusions. These protrusions actively alternated between a straight, relaxed form and a contracted, corkscrew-like one. A peptidoglycan layer was not detected by electron microscopy. The organism forms “fried-egg”-like colonies on MM-X medium. The organism is strictly anaerobic and halophilic and has an optimum temperature for growth of about 30 to 37°C and an optimum pH of about 7. Nitrate and nitrite are reduced; lactate is a fermentation product. The fatty acid profile is dominated by straight saturated and unsaturated chain compounds. Menaquinone 4 is the major respiratory quinone. Phylogenetic analysis demonstrated strain SSD-17B
T
represents a novel and distinct lineage within the radiation of the domain
Bacteria
. The branching position of strain SSD-17B
T
was equidistant to the taxa considered to be representative lineages of the phyla
Firmicutes
and
Tenericutes
(with its sole class
Mollicutes
). The phenotypic and phylogenetic data clearly show the distinctiveness of this unusual bacterium, and we therefore propose that strain SSD-17B
T
(= DSM 18853 = JCM 14575) represents a new genus and a new species, for which we recommend the name
Haloplasma contractile
gen. nov., sp. nov. We are also of the opinion that the organism represents a new order-level taxon, for which we propose the name
Haloplasmatales
.
Quantitative Analysis of Three Hydrogenotrophic Microbial Groups, Methanogenic Archaea, Sulfate-Reducing Bacteria, and Acetogenic Bacteria, within Plaque Biofilms Associated with Human Periodontal Disease
Authors: Vianna M. E. , Holtgraewe S. , Seyfarth I. , Conrads G. , Horz H. P.
Published: March 2008

Human subgingival plaque biofilms are highly complex microbial ecosystems that may depend on H
2
-metabolizing processes. Here we investigated the ubiquity and proportions of methanogenic archaea, sulfate reducers, and acetogens in plaque samples from 102 periodontitis patients. In contrast to the case for 65 healthy control subjects, hydrogenotrophic groups were almost consistently detected in periodontal pockets, with the proportions of methanogens and sulfate reducers being significantly elevated in severe cases. In addition, antagonistic interactions among the three microbial groups indicated that they may function as alternative syntrophic partners of secondary fermenting periodontal pathogens.
Staphylococcus aureus
HrtA Is an ATPase Required for Protection against Heme Toxicity and Prevention of a Transcriptional Heme Stress Response
Authors: Stauff Devin L. , Bagaley Danielle , Torres Victor J. , Joyce Rose , Anderson Kelsi L. , Kuechenmeister Lisa , Dunman Paul M. , Skaar Eric P.
Published: March 2008

During systemic infection,
Staphylococcus aureus
acquires nutrient iron from heme, the cofactor of vertebrate myoglobin and hemoglobin. Upon exposure to heme,
S. aureus
up-regulates the expression of the
h
eme-
r
egulated
t
ransporter, HrtAB. Strains lacking
hrtAB
exhibit increased sensitivity to heme toxicity, and upon heme exposure they elaborate a secreted protein response that interferes with the recruitment of neutrophils to the site of infection. Taken together, these results have led to the suggestion that
hrtAB
encodes an efflux system responsible for relieving the toxic effects of accumulated heme. Here we extend these observations by demonstrating that HrtA is the ATPase component of the HrtAB transport system. We show that HrtA is an Mn
2+
/Mg
2+
-dependent ATPase that functions at an optimal pH of 7.5 and exhibits in vitro temperature dependence uncommon to ABC transporter ATPases. Furthermore, we identify conserved residues within HrtA that are required for in vitro ATPase activity and are essential for the functionality of HrtA in vivo. Finally, we show that heme induces an alteration in the gene expression pattern of
S. aureus
Δ
hrtA
, implying the presence of a novel transcriptional regulatory mechanism responsible for the previously described immunomodulatory characteristics of
hrtA
mutants exposed to heme.