bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Stability and Control of the Bicycle
Authors: Sharp Robin S.
Published: October 2008

After some brief history, a mathematical model of a bicycle that has become a benchmark is described. The symbolic equations of motion of the bicycle are given in two forms and the equations are interpreted, with special reference to stability. The mechanics of autostabilization are discussed in detail. The relationship between design and behavior is shown to be heavily speed-dependent and complex. Using optimal linear preview control theory, rider control of the bicycle is studied. It is shown that steering control by an optimal rider, especially at low speeds, is powerful in comparison with a bicycle’s self-steering. This observation leads to the expectation that riders will be insensitive to variations in design, as has been observed in practice. Optimal preview speed control is also demonstrated. Extensions to the basic treatment of bicycle dynamics in the benchmark case are considered so that the modeling includes more realistic representations of tires, frames, and riders. The implications for stability predictions are discussed and it is shown that the moderate-speed behavior is altered little by the elaborations. Rider control theory is applied to the most realistic of the models considered and the results indicate a strong similarity between the benchmark case and the complex one, where they are directly comparable. In the complex case, steering control by rider-lean-torque is feasible and the results indicate that, when this is combined with steer-torque control, it is completely secondary. When only rider-lean-torque control is possible, extended preview is necessary, high-gain control is required, and the controls are relatively complex. Much that is known about the stability and control of bicycles is collected and explained, together with new material relating to modeling accuracy, bicycle design, and rider control.
Coulomb–Mohr Granular Materials: Quasi-static Flows and the Highly Frictional Limit
Authors: Cox Grant M. , Thamwattana Ngamta , McCue Scott W. , Hill James M.
Published: October 2008

One approach to modeling fully developed shear flow of frictional granular materials is to use a yield condition and a flow rule, in an analogous way to that commonly employed in the fields of metal plasticity and soil mechanics. Typically, the yield condition of choice for granular materials is the Coulomb–Mohr criterion, as this constraint is relatively simple to apply but at the same time is also known to predict stresses that are in good agreement with experimental observations. On the other hand, there is no strong agreement within the engineering and applied mechanics community as to which flow rule is most appropriate, and this subject is still very much open to debate. This paper provides a review of the governing equations used to describe the flow of granular materials subject to the Coulomb–Mohr yield condition, concentrating on the coaxial and double-shearing flow rules in both plane strain and axially symmetric geometries. Emphasis is given to highly frictional materials, which are defined as those granular materials that possess angles of internal friction whose trigonometric sine is close in value to unity. Furthermore, a discussion is provided on the practical problems of determining the stress and velocity distributions in a gravity flow hopper, as well as the stress fields beneath a standing stockpile and within a stable rat-hole.
Microbial Biofilm Voltammetry: Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms
Authors: Marsili Enrico , Rollefson Janet B. , Baron Daniel B. , Hozalski Raymond M. , Bond Daniel R.
Published: October 2008

While electrochemical characterization of enzymes immobilized on electrodes has become common, there is still a need for reliable quantitative methods for study of electron transfer between living cells and conductive surfaces. This work describes growth of thin (<20 μm)
Geobacter sulfurreducens
biofilms on polished glassy carbon electrodes, using stirred three-electrode anaerobic bioreactors controlled by potentiostats and nondestructive voltammetry techniques for characterization of viable biofilms. Routine in vivo analysis of electron transfer between bacterial cells and electrodes was performed, providing insight into the main redox-active species participating in electron transfer to electrodes. At low scan rates, cyclic voltammetry revealed catalytic electron transfer between cells and the electrode, similar to what has been observed for pure enzymes attached to electrodes under continuous turnover conditions. Differential pulse voltammetry and electrochemical impedance spectroscopy also revealed features that were consistent with electron transfer being mediated by an adsorbed catalyst. Multiple redox-active species were detected, revealing complexity at the outer surfaces of this bacterium. These techniques provide the basis for cataloging quantifiable, defined electron transfer phenotypes as a function of potential, electrode material, growth phase, and culture conditions and provide a framework for comparisons with other species or communities.
Volatilization and Precipitation of Tellurium by Aerobic, Tellurite-Resistant Marine Microbes
Authors: Ollivier Patrick R. L. , Bahrou Andrew S. , Marcus Sarah , Cox Talisha , Church Thomas M. , Hanson Thomas E.
Published: October 2008

Microbial resistance to tellurite, an oxyanion of tellurium, is widespread in the biosphere, but the geochemical significance of this trait is poorly understood. As some tellurite resistance markers appear to mediate the formation of volatile tellurides, the potential contribution of tellurite-resistant microbial strains to trace element volatilization in salt marsh sediments was evaluated. Microbial strains were isolated aerobically on the basis of tellurite resistance and subsequently examined for their capacity to volatilize tellurium in pure cultures. The tellurite-resistant strains recovered were either yeasts related to marine isolates of
Rhodotorula
spp. or gram-positive bacteria related to marine strains within the family
Bacillaceae
based on rRNA gene sequence comparisons. Most strains produced volatile tellurides, primarily dimethyltelluride, though there was a wide range of the types and amounts of species produced. For example, the
Rhodotorula
spp. produced the greatest quantities and highest diversity of volatile tellurium compounds. All strains also produced methylated sulfur compounds, primarily dimethyldisulfide. Intracellular tellurium precipitates were a major product of tellurite metabolism in all strains tested, with nearly complete recovery of the tellurite initially provided to cultures as a precipitate. Different strains appeared to produce different shapes and sizes of tellurium containing nanostructures. These studies suggest that aerobic marine yeast and
Bacillus
spp. may play a greater role in trace element biogeochemistry than has been previously assumed, though additional work is needed to further define and quantify their specific contributions.
Synthesis and Uptake of the Compatible Solutes Ectoine and 5-Hydroxyectoine by
Streptomyces coelicolor
A3(2) in Response to Salt and Heat Stresses
Authors: Bursy Jan , Kuhlmann Anne U. , Pittelkow Marco , Hartmann Holger , Jebbar Mohamed , Pierik Antonio J. , Bremer Erhard
Published: October 2008

Streptomyces coelicolor
A3(2) synthesizes ectoine and 5-hydroxyectoine upon the imposition of either salt (0.5 M NaCl) or heat stress (39°C). The cells produced the highest cellular levels of these compatible solutes when both stress conditions were simultaneously imposed. Protection against either severe salt (1.2 M NaCl) or heat stress (39°C) or a combination of both environmental cues could be accomplished by adding low concentrations (1 mM) of either ectoine or 5-hydroxyectoine to
S. coelicolor
A3(2) cultures. The best salt and heat stress protection was observed when a mixture of ectoine and 5-hydroxyectoine (0.5 mM each) was provided to the growth medium. Transport assays with radiolabeled ectoine demonstrated that uptake was triggered by either salt or heat stress. The most effective transport and accumulation of [
14
C]ectoine by
S. coelicolor
A3(2) were achieved when both environmental cues were simultaneously applied. Our results demonstrate that the accumulation of the compatible solutes ectoine and 5-hydroxyectoine allows
S. coelicolor
A3(2) to fend off the detrimental effects of both high salinity and high temperature on cell physiology. We also characterized the enzyme (EctD) required for the synthesis of 5-hydroxyectoine from ectoine, a hydroxylase of the superfamily of the non-heme-containing iron(II)- and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). The gene cluster (
ectABCD
) encoding the enzymes for ectoine and 5-hydroxyectoine biosynthesis can be found in the genome of
S. coelicolor
A3(2),
Streptomyces avermitilis
,
Streptomyces griseus
,
Streptomyces scabiei
, and
Streptomyces chrysomallus
, suggesting that these compatible solutes play an important role as stress protectants in the genus
Streptomyces
.
Distribution of
Crenarchaeota
Representatives in Terrestrial Hot Springs of Russia and Iceland
Authors: Perevalova Anna A. , Kolganova Tatiana V. , Birkeland Nils-Kåre , Schleper Christa , Bonch-Osmolovskaya Elizaveta A. , Lebedinsky Alexander V.
Published: October 2008

Culture-independent (PCR with
Crenarchaeota-
specific primers and subsequent denaturing gradient gel electrophoresis) and culture-dependent approaches were used to study the diversity of
Crenarchaeota
in terrestrial hot springs of the Kamchatka Peninsula and the Lake Baikal region (Russia) and of Iceland. Among the phylotypes detected there were relatives of both cultured (mainly hyperthermophilic) and uncultured
Crenarchaeota
. It was found that there is a large and diverse group of uncultured
Crenarchaeota
that inhabit terrestrial hot springs with moderate temperatures (55 to 70°C). Two of the lineages of this group were given phenotypic characterization, one as a result of cultivation in an enrichment culture and another one after isolation of a pure culture, “
Fervidococcus fontis
,” which proved to be a moderately thermophilic, neutrophilic (optimum pH of 6.0 to 7.5), anaerobic organotroph.
International Comparison of Clinical, Bovine, and Environmental
Escherichia coli
O157 Isolates on the Basis of Shiga Toxin-Encoding Bacteriophage Insertion Site Genotypes
Authors: Whitworth Joshua H. , Fegan Narelle , Keller Jasmin , Gobius Kari S. , Bono James L. , Call Douglas R. , Hancock Dale D. , Besser Thomas E.
Published: October 2008

Escherichia coli
O157:H7 genotypes in the bovine reservoir may differ in virulence. The proportion of clinical genotypes among cattle isolates was weakly (
P
= 0.054) related to the international incidence of
E. coli
O157:H7-associated hemolytic-uremic syndrome, varied among clinical isolates internationally, and also differed along the putative cattle-hamburger-clinical case transmission chain.
Widespread Distribution and Identification of Eight Novel Microcystins in Antarctic Cyanobacterial Mats
Authors: Wood Susanna A. , Mountfort Doug , Selwood Andrew I. , Holland Patrick T. , Puddick Jonathan , Cary S. Craig
Published: October 2008

The microcystin (MC) content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes, and hydroterrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys, Wright, Victoria, Miers, and Marshall. Enzyme-linked immunosorbent assays (ELISAs), liquid chromatography-mass spectrometry (LC-MS), and protein phosphatase 2A (PP-2A) inhibition assays resulted in the identification of low levels (1 to 16 mg/kg [dry weight]) of MCs in all samples. A plot of indicative potencies of MCs (PP-2A inhibition assay/ELISA ratio) versus total MCs (ELISA) showed a general decrease in potency, as total MC levels increased, and a clustering of values from discrete geographic locations. LC-tandem MS analysis on selected samples identified eight novel MC congeners. The low-energy collisional activation spectra were consistent with variants of [
d
-Asp
3
] MC-RR and [
d
-Asp
3
] MC-LR containing glycine [Gly
1
] rather than alanine and combinations of homoarginine [hAr
2
] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda
5
] substitutions.
Nostoc
sp. was identified as a MC producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase domain of the
mcyE
gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general, samples from the same geographic location tended to cluster together. ARISA also enabled the putative identification of the MC-producing
Nostoc
sp. from multiple samples.
Quantification of Diatom and Dinoflagellate Biomasses in Coastal Marine Seawater Samples by Real-Time PCR
Authors: Godhe Anna , Asplund Maria E. , Härnström Karolina , Saravanan V. , Tyagi Anuj , Karunasagar Indrani
Published: October 2008

Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.
Acanthamoeba castellanii
Promotes the Survival of
Vibrio parahaemolyticus
Authors: Laskowski-Arce Michelle A. , Orth Kim
Published: October 2008

Vibrio parahaemolyticus
is a food-borne pathogen that naturally inhabits both marine and estuarine environments. Free-living protozoa exist in similar aquatic environments and function to control bacterial numbers by grazing on free-living bacteria. Protozoa also play an important role in the survival and spread of some pathogenic species of bacteria. We investigated the interaction between the protozoan
Acanthamoeba castellanii
and the bacterium
Vibrio parahaemolyticus
. We found that
Acanthamoeba castellanii
does not prey on
Vibrio parahaemolyticus
but instead secretes a factor that promotes the survival of
Vibrio parahaemolyticus
in coculture. These studies suggest that protozoa may provide a survival advantage to an extracellular pathogen in the environment.
P
BAD
-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in
Pseudomonas
and
Burkholderia
spp. and Other Bacteria
Authors: Qiu Dongru , Damron F. Heath , Mima Takehiko , Schweizer Herbert P. , Yu Hongwei D.
Published: October 2008

We report the construction of a series of
Escherichia
-
Pseudomonas
broad-host-range expression vectors utilizing the P
BAD
promoter and the
araC
regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.
Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of
Manduca sexta
(Sphingidae, Lepidoptera, Insecta)
Authors: Brinkmann Nicole , Martens Rainer , Tebbe Christoph C.
Published: October 2008

Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCR-single-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of
Manduca sexta
bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a
13
CO
2
-enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an
Enterococcus
relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a
Citrobacter sedlakii
relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A
Burkholderia
relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (
Enterobacter
sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.
Phenotype-Specific Bacterial Communities in the Cold-Water Coral
Lophelia pertusa
(Scleractinia) and Their Implications for the Coral's Nutrition, Health, and Distribution
Authors: Neulinger Sven C. , Järnegren Johanna , Ludvigsen Martin , Lochte Karin , Dullo Wolf-Christian
Published: October 2008

The pseudocolonial coral
Lophelia pertusa
(Scleractinia, Caryophylliidae) is a eurybathic, stenothermal cosmopolitan cold-water species. It occurs in two color varieties, white and red.
L. pertusa
builds vast cold-water coral reefs along the continental margins, which are among the most diverse deep-sea habitats. Microbiology of
L. pertusa
has been in scientific focus for only a few years, but the question of whether the coral holds a host-specific bacterial community has not been finally answered. Bacteria on coral samples from the Trondheimsfjord (Norway) were characterized by the culture-independent 16S rRNA gene-based techniques terminal restriction fragment length polymorphism and sequence analysis.
L. pertusa
revealed a high microbial richness. Clone sequences were dominated by members of the
Alpha
- and
Gammaproteobacteria
. Other abundant taxa were
Bacteroidetes
,
Actinobacteria
,
Verrucomicrobia
,
Firmicutes
, and
Planctomycetes
. The bacterial community of
L. pertusa
not only differed conspicuously from that of the environment but also varied with both the location and color variety of its host. Therefore, the microbial colonization cannot be termed “specific” sensu stricto. However, similarities to other coral-bacterium associations suggest the existence of “cold-water coral-specific” bacterial groups sensu lato.
L. pertusa
-associated bacteria appear to play a significant role in the nutrition of their host by degradation of sulfur compounds, cellulose, chitin, and end products of the coral's anaerobic metabolism. Some coral-associated microbes were regarded as opportunistic pathogens. Dominance of mixotrophic members of the
Rhodobacteraceae
in white
L. pertusa
could explain the wider dispersal of this phenotype by supplementary nutrition.
Generation of
Bradyrhizobium japonicum
Mutants with Increased N
2
O Reductase Activity by Selection after Introduction of a Mutated
dnaQ
Gene
Authors: Itakura Manabu , Tabata Kazufumi , Eda Shima , Mitsui Hisayuki , Murakami Kiriko , Yasuda Junichi , Minamisawa Kiwamu
Published: October 2008

We obtained two beneficial mutants of
Bradyrhizobium japonicum
USDA110 with increased nitrous oxide (N
2
O) reductase (N
2
OR) activity by introducing a plasmid containing a mutated
B. japonicum dnaQ
gene (pKQ2) and performing enrichment culture under selection pressure for N
2
O respiration. Mutation of
dnaQ
, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in
Escherichia coli
. pKQ2 introduction into
B. japonicum
USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N
2
O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N
2
OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N
2
OR activity of the wild-type USDA110. Although two mutants had a weak or null
fix
phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.
Thiol-Independent Action of Mitochondrial Thioredoxin To Support the Urea Cycle of Arginine Biosynthesis in
Schizosaccharomyces pombe
Authors: Song Ji-Yoon , Kim Kyoung-Dong , Roe Jung-Hye
Published: October 2008

Thioredoxins usually perform a role as a thiol-disulfide oxidoreductase using their active-site cysteines. The fission yeast
Schizosaccharomyces pombe
contains two thioredoxins: Trx1 for general stress protection and Trx2 for mitochondrial functions. The
Δtrx2
mutant grows as well as the wild type on complex media containing glucose. However, on nonfermentable carbon source such as glycerol, the mutant did not grow, indicating a defect in mitochondrial function. The mutant also exhibited auxotrophy for arginine and cysteine on minimal medium. In order to find the reason for the unexpected arginine auxotrophy, we searched for multicopy suppressors and found that the
arg3
+
gene encoding ornithine carbamoyltransferase (OCTase) in the urea cycle of the arginine biosynthetic pathway rescued the arginine auxotrophy. The levels of
arg3
+
transcript, Arg3 protein, and OCTase activity were all decreased in
Δtrx2
. Through immunocoprecipitation, we observed a direct interaction between Trx2 and Arg3 in cell extracts. The mutant forms of Trx2 lacking either one or both of the active site cysteines through substitution to serines also rescued the arginine auxotrophy and restored the decreased OCTase activity. They also rescued the growth defect of
Δtrx2
on glycerol medium. This contrasts with the thiol-dependent action of overproduced Trx2 in complementing glutathione reductase. Therefore, Trx2 serves multiple functions in mitochondria, protecting mitochondrial components against thiol-oxidative damage as a thiol-disulfide oxidoreductase, and supporting urea cycle and respiration in mitochondria in a manner independent of active site thiols.
C2 Domain Protein MIN1 Promotes Eyespot Organization inChlamydomonas reinhardtii
Authors: Mittelmeier Telsa M. , Berthold Peter , Danon Avihai , Lamb Mary Rose , Levitan Alexander , Rice Michael E. , Dieckmann Carol L.
Published: October 2008

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green algaChlamydomonas reinhardtiirequires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast.min1(mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. TheMIN1gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly inChlamydomonas. Inmin1null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth ofmin1cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.
Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes
Authors: Chandler Julian , Vandoros Anthula V. , Mozeleski Brian , Klingbeil Michele M.
Published: October 2008

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from
Trypanosoma brucei
with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic
T. brucei
. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol γ) for replication of mitochondrial DNA,
T. brucei
requires at least three to maintain the complex kDNA network.
In Vitro CpG Methylation Increases the Transformation Efficiency ofBorrelia burgdorferiStrains Harboring the Endogenous Linear Plasmid lp56
Authors: Chen Qiang , Fischer Joshua R. , Benoit Vivian M. , Dufour Nicholas P. , Youderian Philip , Leong John M.
Published: October 2008

Borrelia burgdorferiis the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains ofB. burgdorferiexhibit extremely low transformation efficiencies—so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poorB. burgdorferitransformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that forB. burgdorferistrains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation ofB. burgdorferiwas exemplified by the ease of plasmid complementation of aB. burgdorferiB31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kanr) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, theB. burgdorferiB31 A3 BBK32::Kanrmutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.
Flexibility of
Vibrio cholerae
ToxT in Transcription Activation of Genes Having Altered Promoter Spacing
Authors: Bellair Michelle , Withey Jeffrey H.
Published: October 2008

Cholera, a severe diarrheal disease, is caused by ingestion of the gram-negative bacterium
Vibrio cholerae
. Expression of
V. cholerae
virulence factors is highly regulated at the transcriptional and posttranscriptional levels by a complex network of proteins and small noncoding RNAs. The direct activator of transcription of most
V. cholerae
virulence genes is the ToxT protein. ToxT binds to a 13-bp sequence, the toxbox, located upstream of genes in its regulon. However, the organization of toxboxes relative to each other and to the core promoter elements at different genes varies dramatically. At different ToxT-activated genes a single toxbox may be necessary and sufficient for full activation, or pairs of toxboxes organized as either inverted or direct repeats may be required for full activation. Although all toxboxes are located at positions consistent with a class I promoter architecture, the locations of toxboxes relative to the transcription start site also vary from gene to gene. To further assess the ability of ToxT to activate transcription from different configurations relative to the core promoter elements, we constructed promoter-
lacZ
fusions having altered spacing both between toxbox pairs and between the promoter-proximal toxbox and the −35 box at five different ToxT-activated promoters. Our results suggest that that ToxT has remarkable flexibility in its positioning as a transcription activator and that different interactions between ToxT and RNA polymerase occur during transcription activation of promoters having different toxbox configurations.
The Global Repressor SugR Controls Expression of Genes of Glycolysis and of the
l
-Lactate Dehydrogenase LdhA in
Corynebacterium glutamicum
Authors: Engels Verena , Lindner Steffen N. , Wendisch Volker F.
Published: October 2008

The transcriptional regulator SugR from
Corynebacterium glutamicum
represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of
sugR
not only perturbed the growth of
C. glutamicum
on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (
pfkA
), fructose-1,6-bisphosphate aldolase (
fba
), enolase (
eno
), pyruvate kinase (
pyk
), and NAD-dependent
l
-lactate dehydrogenase (
ldhA
). The deletion of
sugR
resulted in increased mRNA levels of
eno
,
pyk
, and
ldhA
in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of
sugR
led to increased LdhA activity under aerobic and under oxygen deprivation conditions,
l
-lactate production by
C. glutamicum
was determined. The overexpression of
sugR
reduced
l
-lactate production by about 25%, and
sugR
deletion increased
l
-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative
l
-lactate dehydrogenase in
C. glutamicum
.
Dimethylsulfoniopropionate-Dependent Demethylase (DmdA) from
Pelagibacter ubique
and
Silicibacter pomeroyi
Authors: Reisch Chris R. , Moran Mary Ann , Whitman William B.
Published: October 2008

The ubiquitous algal metabolite dimethylsulfoniopropionate (DMSP) is a major source of carbon and reduced sulfur for marine bacteria. Recently, the enzyme responsible for the demethylation of DMSP, designated DmdA, was identified, and homologs were found to be common in marine bacterioplankton cells. The recombinant DmdA proteins from the cultured marine bacteria
Pelagibacter ubique
HTCC1062 and
Silicibacter pomeroyi
DSS-3 were purified with a three-step procedure using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatographies. The
P. ubique
enzyme possessed an
M
r
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 38,500. Under nondenaturing conditions, the
M
r
was 68,000, suggesting that the enzyme was likely to be a dimer. The purified enzyme exhibited strict substrate specificity for DMSP, as DmdA from both
S. pomeroyi
and
P. ubique
possessed no detectable demethylase activity with glycine betaine, dimethyl glycine, methylmercaptopropionate, methionine, or dimethylsulfonioacetate. Less than 1% activity was found with dimethylsulfoniobutanoate and dimethylsulfoniopentanoate. The apparent

K
m

s for DMSP were 13.2 ± 2.0 and 5.4 ± 2.3 mM for the
P. ubique
and
S. pomeroyi
enzymes, respectively. In cell extracts of
S. pomeroyi
DSS-3, the apparent

K
m

for DMSP was 8.6 ± 1.2 mM, similar to that of purified recombinant DmdA. The intracellular concentration of DMSP in chemostat-grown
S. pomeroyi
DSS-3 was 70 mM. These results suggest that marine bacterioplankton may actively accumulate DMSP to osmotically significant concentrations that favor near-maximal rates of DMSP demethylation activity.
Bacterial Flagella Are Firmly Anchored
Authors: Darnton Nicholas C. , Berg Howard C.
Published: October 2008

There are mutants of
Salmonella enterica
(with mutations in
fliF
and
fliL
) that shed flagella when they are swimming in a viscous medium or on the surface of soft agar. Filaments with hooks and the distal rod segment FlgG are recovered. We tried to extract flagellar filaments from such cells by pulling on them with an optical trap but failed, even when we used forces large enough to straighten the filaments. Thus, flagella are firmly anchored.
Site-Directed Mutagenesis Reveals Putative Substrate Binding Residues in the
Escherichia coli
RND Efflux Pump AcrB
Authors: Bohnert Jürgen A. , Schuster Sabine , Seeger Markus A. , Fähnrich Eva , Pos Klaas M. , Kern Winfried V.
Published: October 2008

The
Escherichia coli
multidrug efflux pump protein AcrB has recently been cocrystallized with various substrates, suggesting that there is a phenylalanine-rich binding site around F178 and F615. We found that F610A was the point mutation that had the most significant impact on substrate MICs, while other targeted mutations, including conversion of phenylalanines 136, 178, 615, 617, and 628 to alanine, had smaller and more variable effects.
Genetic Analysis ofVibrio choleraeMonolayer Formation Reveals a Key Role for ΔΨ in the Transition to Permanent Attachment
Authors: Van Dellen Katrina L. , Houot Laetitia , Watnick Paula I.
Published: October 2008

A bacterial monolayer biofilm is a collection of cells attached to a surface but not to each other. Monolayer formation is initiated when a bacterial cell forms a transient attachment to a surface. While some transient attachments are broken, others transition into the permanent attachments that define a monolayer biofilm. In this work, we describe the results of a large-scale, microscopy-based genetic screen forVibrio choleraemutants that are defective in formation of a monolayer biofilm. This screen identified mutations that alter both transient and permanent attachment. Transient attachment was somewhat slower in the absence of flagellar motility. However, flagellar mutants eventually formed a robust monolayer. In contrast, in the absence of the flagellar motor, monolayer formation was severely impaired. A number of proteins that modulate theV. choleraeion motive force were also found to affect the transition from transient to permanent attachment. Using chemicals that dissipate various components of the ion motive force, we discovered that dissipation of the membrane potential (ΔΨ) completely blocks the transition from transient to permanent attachment. We propose that as a bacterium approaches a surface, the interaction of the flagellum with the surface leads to transient hyperpolarization of the bacterial cell membrane. This, in turn, initiates the transition to permanent attachment.
The Biosynthetic Pathway for Synechoxanthin, an Aromatic Carotenoid Synthesized by the Euryhaline, Unicellular Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Graham Joel E. , Bryant Donald A.
Published: October 2008

The euryhaline, unicellular cyanobacterium
Synechococcus
sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (χ,χ-caroten-18,18′-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (χ,χ-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in
Synechococcus
sp. strain PCC 7002 to verify their function. The
cruE
gene (SYNPCC7002_A1248) encodes β-carotene desaturase/methyltransferase, which converts β-carotene to renierapurpurin. The
cruH
gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18′ methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.
Loss of RNase R Induces Competence Development in
Legionella pneumophila
Authors: Charpentier Xavier , Faucher Sébastien P. , Kalachikov Sergey , Shuman Howard A.
Published: October 2008

RNase R is a processive 3′-5′ exoribonuclease with a high degree of conservation in prokaryotes. Although some bacteria possess additional hydrolytic 3′-5′ exoribonucleases such as RNase II, RNase R was found to be the only predicted one in the facultative intracellular pathogen
Legionella pneumophila
. This provided a unique opportunity to study the role of RNase R in the absence of an additional RNase with similar enzymatic activity. We investigated the role of RNase R in the biology of
Legionella pneumophila
under various conditions and performed gene expression profiling using microarrays. At optimal growth temperature, the loss of RNase R had no major consequence on bacterial growth and had a moderate impact on normal gene regulation. However, at a lower temperature, the loss of RNase R had a significant impact on bacterial growth and resulted in the accumulation of structured RNA degradation products. Concurrently, gene regulation was affected and specifically resulted in an increased expression of the competence regulon. Loss of the exoribonuclease activity of RNase R was sufficient to induce competence development, a genetically programmed process normally triggered as a response to environmental stimuli. The temperature-dependent expression of competence genes in the
rnr
mutant was found to be independent of previously identified competence regulators in
Legionella pneumophila
. We suggest that a physiological role of RNase R is to eliminate structured RNA molecules that are stabilized by low temperature, which in turn may affect regulatory networks, compromising adaptation to cold and thus resulting in decreased viability.
Structure of
l
-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic
l
-Ascorbate Utilization Pathway of
Escherichia coli
: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold
Authors: Shi Rong , Pineda Marco , Ajamian Eunice , Cui Qizhi , Matte Allan , Cygler Miroslaw
Published: October 2008

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of
l
-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce
l
-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (
l
-xylulose-5-phosphate 3-epimerase) and UlaF (
l
-ribulose-5-phosphate 4-epimerase), yielding
d
-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from
Escherichia coli
O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn
2+
, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/α1 loop and α3′ helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the
l
-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.
Discovering Functional Novelty in Metagenomes: Examples from Light-Mediated Processes
Authors: Singh Amoolya H. , Doerks Tobias , Letunic Ivica , Raes Jeroen , Bork Peer
Published: October 2008

The emerging coverage of diverse habitats by metagenomic shotgun data opens new avenues of discovering functional novelty using computational tools. Here, we apply three different concepts for predicting novel functions within light-mediated microbial pathways in five diverse environments. Using phylogenetic approaches, we discovered two novel deep-branching subfamilies of photolyases (involved in light-mediated repair) distributed abundantly in high-UV environments. Using neighborhood approaches, we were able to assign seven novel functional partners in luciferase synthesis, nitrogen metabolism, and quorum sensing to BLUF domain-containing proteins (involved in light sensing). Finally, by domain analysis, for RcaE proteins (involved in chromatic adaptation), we predict 16 novel domain architectures that indicate novel functionalities in habitats with little or no light. Quantification of protein abundance in the various environments supports our findings that bacteria utilize light for sensing, repair, and adaptation far more widely than previously thought. While the discoveries illustrate the opportunities in function discovery, we also discuss the immense conceptual and practical challenges that come along with this new type of data.
Oxidant-Responsive Induction of the
suf
Operon, Encoding a Fe-S Assembly System, through Fur and IscR in
Escherichia coli
Authors: Lee Kyung-Chang , Yeo Won-Sik , Roe Jung-Hye
Published: October 2008

The
suf
operon encoding a Fe-S assembly system is induced by peroxides through activators OxyR and IscR in
Escherichia coli
. For apo-IscR to bind, oxidation-mediated dissociation of Fur is required. Therefore, a peroxide-responsive signal is transduced through OxyR, IscR, and Fur to achieve oxidation-sensitive and maximal induction of this operon.
Avoidance of smoking: the impact of warning labels in Brazil
Authors: Nascimento B E M , Oliveira L , Vieira A S , Joffily M , Gleiser S , Pereira M G , Cavalcante T , Volchan E
Published: October 2008

Background:

Research on human emotion shows that pictures drive the activity of specialised brain networks affecting attitude and behaviour. Pictorial warnings on cigarette packages are considered one of the most effective ways to convey information on the health consequences of smoking. However, few studies have evaluated the effectiveness of warning labels to elicit avoidance of smoking.

Objectives:

To investigate the impact of pictorial health warnings conveyed by the Brazilian tobacco control programme through a well-established psychometric tool designed for studies on emotion and behaviour.

Methods:

Graphic Brazilian cigarette warnings labels were evaluated. They consisted of the two sets of warning pictures displayed in 2002–4 (n = 9) and 2004–8 (n = 10). Pleasant, unpleasant and neutral pictures selected from a standard catalogue were used as controls. Undergraduate students (n = 212, 18% smokers) evaluated the emotional content of each picture in two affective dimensions: hedonic valence and arousal. Participants were not provided with the sources of distinction between control and warning pictures.

Results:

The judgements of hedonic content of the warning pictures ranged from neutral to very unpleasant. None was classified as highly arousing. Smokers judged warning pictures representing people smoking significantly more pleasant than pictures without smoking scenes, and significantly more so than non-smokers. No significant differences between smokers and non-smokers were found for warning pictures without these smoking scenes.

Conclusion:

Previous studies have shown that the most threatening and arousing pictures prompt the greatest evidence of defensive activation. Emotional ratings of Brazilian warning pictures described them as unpleasant but moderately arousing. To intensify avoidance of the packages, future graphic warnings should therefore generate more arousal. The ratings for the Brazilian warning pictures indicated that, except for those depicting people smoking, judgements by smokers and non-smokers were similar, suggesting a potential applicability in both prevention and cessation. Smoking cues, however, should be avoided.
Timing of adenosine 2A receptor stimulation relative to reperfusion has differential effects on infarct size and cardiac function as assessed in mice by MRI
Authors: Yang Zequan , Linden Joel , Berr Stuart S. , Kron Irving L. , Beller George A. , French Brent A.
Published: October 2008

The activation of adenosine 2A receptors before reperfusion following coronary artery occlusion reduces infarct size and improves ejection fraction (EF). In this study, we examined the effects of delaying treatment with the adenosine 2A receptor agonist ATL146e (ATL) until 1 h postreperfusion. The infarct size and EF were serially assessed by gadolinium-diethylenetriaminepentaacetic acid-enhanced MRI in C57BL/6 mice at 1 and 24 h postreperfusion. The infarct size was also assessed by 2,3,5-triphenyltetrazolium chloride staining at 24 h. Mice were treated with ATL (10 μg/kg ip) either 2 min before reperfusion (early ATL) or 1 h postreperfusion (late ATL) following the 45-min coronary occlusion. The two methods used to assess infarct size at 24 h postreperfusion (MRI and 2,3,5-triphenyltetrazolium chloride) showed an excellent correlation ( R = 0.96). The risk region, determined at 24 h postreperfusion, was comparable between the control and ATL-treated groups. The infarct size by MRI at 1 versus 24 h postreperfusion was 25 ± 1 vs. 26 ± 1% of left ventricular mass (means ± SE) in control mice, 16 ± 2 versus 17 ± 2% in early-ATL mice, and 24 ± 2 versus 25 ± 2% in late-ATL mice (intragroup, P = not significant; and intergroup, early ATL vs. control or late ATL, P < 0.05). EF was reduced in control mice but was largely preserved between 1 and 24 h in both early-ATL and late-ATL mice ( P < 0.05). In conclusion, after coronary occlusion in mice, the extent of myocellular death due to ischemia-reperfusion injury is 95% complete within 1 h of reperfusion. The infarct size was significantly reduced by ATL when given just before reperfusion, but not 1 h postreperfusion. Either treatment window helped preserve the EF between 1 and 24 h postreperfusion.