bluuebunny/crossref_metadata_2025 huggingface dataset:
Bubble Dynamics Observed in a Gas-Liquid Venturi Flow
Authors: Nishi Wataru , Nogami Masanori , Takahira Hiroyuki
Published: June 2012

The present study deals with the experiments for the gas-liquid two-phase flow inside an acrylic Venturi tube using a high-speed video camera. Some interesting phenomena on the bubble dynamics are observed in the tube. First, the volume and surface oscillations of two interacting bubbles are observed in converging section of the tube when one bubble enters the throat. The volume oscillation of the bubble that enters the throat is caused by the detachment of the tip of the downstream surface of the bubble. The pressure wave irradiated from the bubble that enters the throat induces the volume and surface oscillations of the bubble that remains at the converging section. The parametric excitation is the reason for the surface oscillations. Second, the bubble deformations at the throat in a Venturi or a converging tube are investigated. The experiments show that two kinds of liquid jets are formed on the bubble surface; one is a forward jet that develops from the upstream surface to the downstream surface and the other is a counter jet in which the direction of the jet is opposite to the forward jet. It is shown that the counter jet occurs only when the distance between two bubbles in the throat is sufficiently short. The interactions between two bubbles cause the counter jet. It is also shown that the velocity of the forward jet becomes faster when the bubble is pinched off more upstream in the converging section. Finally, the propagations of the pressure waves are measured with pressure transducers. The impulsive pressure associated with the collapse of cavitation bubble cloud is measured when a bubble enters the throat of the tube. Also, the propagation speed of pressure waves is evaluated with the cross-correlation function. The results show that the propagation speed and damping of the pressure waves are dependent on the number density of bubbles at the downstream part in the tube.
Observation and Scaling of Tip Vortex Cavitation on Elliptical Hydrofoils
Authors: Nagaya Shigeki , Kimoto Risa , Naganuma Kenji , Mori Takayuki
Published: June 2012

Experimental study on tip vortex cavitation (TVC) was carried out for elliptical hydrofoils with various chord lengths. The purpose of the experiment was to clarify the influences of Reynolds number and water quality on tip vortex cavitation. Experiments were made in a large cavitation tunnel of the Naval Systems Research Center, TRDI/Ministry of Defense Japan. The elliptical hydrofoils tested were NACA 0012 cross section with chord lengths of 500mm, 250mm and 50mm. Reynolds number based on hydrofoil chord length was 2×105 < ReC < 7.4×106. Water quality of the tunnel was characterized by air content and nuclei distribution. Air content of the tunnel was varied between 30% and 80%. Nuclei distribution was measured by a cavitation susceptibility meter (CSM) with center-body venturi. Cavitation inception was determined from high speed video observation. A standard formula, (σL/σS) = (ReL/ReS)n, was applied for the scaling. In the present study, exponent of the scaling law n was found to be 0.2 < n < 0.4. High speed video observation showed that the process of the TVC inception strongly depends on water quality. In the experiments, unsteady behaviors of TVC were also investigated. Strong interactions between sheet cavitation and TVC were observed.
An Investigation of Cavitation and Suction Vortices Behavior in Pump Sump
Authors: Kang Won-Tae , Yu Ki Han , Lee Seung Yeob , Shin Byeong Rog
Published: June 2012

A numerical and an experimental investigation on a suction vortices including cavitation, free vortices and subsurface vortices behavior in the model sump system with multi-intakes is performed at several flow rates and water levels. A test model sump and piping system were designed based on Froude similitude for the prototype of the recommended structure layout by HI-9.8 American National Standard for Pump Intake Design of the Hydraulic Institute. An experiment is performed according to the sump model test procedure of Hyosung Goodsprings, Inc. A numerical analysis of three dimensional multiphase flows through the model sump is performed by using the finite volume method of the CFX code with multi-block structured grid systems. A k-ω Shear Stress Transport turbulence model and the Rayleigh-Plesset cavitation model are used for solving turbulence cavitating flow. Several types of free surface and submerged vortex which occurs with each different water level are identified through the experimental investigation. From the numerical analysis, the vortices are reproduced and their formation, growing, shedding and detailed vortex structures are investigated. To reduce abnormal vortices, an anti-vortex device is considered and its effect is investigated and discussed.
Development of Gating Nanopores for Next-Generation DNA Sequencing Using Mechanically Controllable Break Junctions
Authors: Taniguchi Masateru , Kawai Tomoji
Published: June 2012

We have developed two types of devices—vertical and parallel—for incorporating a microfluidic channel into a gating nanopore. The vertical device consists of a single nanogap electrode with a nanopore perpendicular to the surface of a silicon substrate. The parallel device is similar, except the nanopore is parallel to the surface of the substrate. Furthermore, while the vertical device was fabricated using nanofabrication technologies, the parallel device was fabricated using a mechanically controllable break junction that enables atomic-level control of the electrode gap; hence, measurement of single atoms and molecules is possible. Both devices can identify single gold nanoparticles passing through them by measuring the strength of their electrical signals. The parallel device can also identify the individual nucleotides in a DNA molecule.
Development of an Electron Scattering Model to Detect Differences in DNA Base Molecules
Authors: Nishioka Yuki , Doi Kentaro , Kawano Satoyuki
Published: June 2012

In recent, novel technologies which apply bio-macromolecules to bio-nanodevices attract much attention. Particularly, DNAs have several desirable characteristics: complementary base pairs, self assembly, and electric conductivity. It is expected that high-speed DNA sequencers can be developed by using these specific characteristics of DNAs. In the present study, we develop a theoretical model to analyze the difference of DNA base molecules, in which electron scattering is simulated based on classical electrodynamics and scattering angles are evaluated. Consequently, it is found that scattering angles of the scattered electrons are clearly different from each other.
Improvement of Proton Conduction in PEFC by Applying External Perturbations
Authors: Hashizume Hiroki , Doi Kentaro , Kawano Satoyuki
Published: June 2012

In fuel cell technologies, there are some problems which should be overcome rapidly. For example, it is known that voltage losses in polymer electrolyte fuel cells are serious problems. However, drastic solutions have not been found out yet. In the present study, we experimentally try to improve the energy conversion efficiency, focusing on ion currents in electrode membrane assemblies (MEAs). Additional Ag electrodes are fabricated on an MEA and their effects on proton flows are investigated. Consequently, it is found that electric voltage losses can be suppressed by electric or ion currents parallel to the proton flow. This result suggests that the external perturbations can be effective to protons and oxygen molecules on their reactions and on the power generation efficiency.
Host-Gut Microbiota Metabolic Interactions
Authors: Nicholson Jeremy K. , Holmes Elaine , Kinross James , Burcelin Remy , Gibson Glenn , Jia Wei , Pettersson Sven
Published: June 2012

The composition and activity of the gut microbiota codevelop with the host from birth and is subject to a complex interplay that depends on the host genome, nutrition, and life-style. The gut microbiota is involved in the regulation of multiple host metabolic pathways, giving rise to interactive host-microbiota metabolic, signaling, and immune-inflammatory axes that physiologically connect the gut, liver, muscle, and brain. A deeper understanding of these axes is a prerequisite for optimizing therapeutic strategies to manipulate the gut microbiota to combat disease and improve health.
Two Genera of Magnetococci with Bean-like Morphology from Intertidal Sediments of the Yellow Sea, China
Authors: Zhang Wen-Yan , Zhou Ke , Pan Hong-Miao , Yue Hai-Dong , Jiang Ming , Xiao Tian , Wu Long-Fei
Published: June 2012

Magnetotactic bacteria have the unique capacity of being able to swim along geomagnetic field lines. They are Gram-negative bacteria with diverse morphologies and variable phylogenetic relatedness. Here, we describe a group of uncultivated marine magnetococci collected from intertidal sediments of Huiquan Bay in the Yellow Sea. They were coccoid-ovoid in morphology, with an average size of 2.8 ± 0.3 μm by 2.0 ± 0.2 μm. Differential interference contrast microscopy, fluorescence microscopy, and transmission electron microscopy revealed that each cell was apparently composed of two hemispheres. The cells synthesized iron oxide-type magnetosomes that clustered on one side of the cell at the interface between the two hemispheres. In some cells two chains of magnetosomes were observed across the interface. Each cell had two bundles of flagella enveloped in a sheath and displayed north-seeking helical motion. Two 16S rRNA gene sequences having 91.8% identity were obtained, and their authenticity was confirmed by fluorescence
in situ
hybridization. Phylogenetic analysis revealed that the magnetococci are affiliated with the
Alphaproteobacteria
and are most closely related to two uncultured magnetococci with sequence identities of 92.7% and 92.4%, respectively. Because they display a >7% sequence divergence to all bacteria reported, the bean-like magnetococci may represent two novel genera.
Involvement of RpoN in Regulating Bacterial Arsenite Oxidation
Authors: Kang Yoon-Suk , Bothner Brian , Rensing Christopher , McDermott Timothy R.
Published: June 2012

In this study with the model organism
Agrobacterium tumefaciens
, we used a combination of
lacZ
gene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of As
III
oxidation. A deletion mutation that removed the RpoN binding site from the
aioBA
promoter and an
aacC3
(gentamicin resistance) cassette insertional inactivation of the
rpoN
coding region eliminated
aioBA
expression and As
III
oxidation, although
rpoN
expression was not related to cell exposure to As
III
. Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters for
aioB
,
phoB1
,
pstS1
,
dctA
,
glnA
,
glnB
, and
flgB
that were examined by using qualitative RT-PCR and
lacZ
reporter fusions to assess the relative contribution of RpoN to their transcription. The expressions of
aioB
and
dctA
in the wild-type strain were considerably enhanced in cells exposed to As
III
, and both genes were silent in the
rpoN
::
aacC3
mutant regardless of As
III
. The expression level of
glnA
was not influenced by As
III
but was reduced (but not silent) in the
rpoN
::
aacC3
mutant and further reduced in the mutant under N starvation conditions. The
rpoN
::
aacC3
mutation had no obvious effect on the expression of
glnB
,
pstS1
,
phoB1
, or
flgB
. These experiments provide definitive evidence to document the requirement of RpoN for As
III
oxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by As
III
or by this sigma factor.
Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples
Authors: Pires Ana C. C. , Cleary Daniel F. R. , Almeida Adelaide , Cunha Ângela , Dealtry Simone , Mendonça-Hagler Leda C. S. , Smalla Kornelia , Gomes Newton C. M.
Published: June 2012

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed
Archaea
in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of
Archaea
in mangrove bulk sediment and the rhizospheres of two mangrove trees,
Rhizophora mangle
and
Laguncularia racemosa
, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as
Archaea
, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the
L. racemosa
rhizosphere, intermediate in bulk sediment, and the lowest in the
R. mangle
rhizosphere. This study shows that rhizosphere microhabitats of
R. mangle
and
L. racemosa
, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.
A Specific Mutation in the Promoter Region of the Silent
cel
Cluster Accounts for the Appearance of Lactose-Utilizing Lactococcus lactis MG1363
Authors: Solopova Ana , Bachmann Herwig , Teusink Bas , Kok Jan , Neves Ana Rute , Kuipers Oscar P.
Published: June 2012

The
Lactococcus lactis
laboratory strain MG1363 has been described to be unable to utilize lactose. However, in a rich medium supplemented with lactose as the sole carbon source, it starts to grow after prolonged incubation periods. Transcriptome analyses showed that
L. lactis
MG1363 Lac
+
cells expressed
celB
, encoding a putative cellobiose-specific phosphotransferase system (PTS) IIC component, which is normally silent in MG1363 Lac
−
cells. Nucleotide sequence analysis of the
cel
cluster of a Lac
+
isolate revealed a change from one of the guanines to adenine in the promoter region. We showed here that one particular mutation, taking place at increased frequency, accounts for the lactose-utilizing phenotype occurring in MG1363 cultures. The G-to-A transition creates a −10 element at an optimal distance from the −35 element. Thus, a fully active promoter is created, allowing transcription of the otherwise cryptic cluster. Nuclear magnetic resonance (NMR) spectroscopy results show that MG1363 Lac
+
uses a novel pathway of lactose utilization.
Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures
Authors: Lindström Miia , Dahlsten Elias , Söderholm Henna , Selby Katja , Somervuo Panu , Heap John T. , Minton Nigel P. , Korkeala Hannu
Published: June 2012

The role of the two-component system (TCS) CBO0366/CBO0365 in the cold shock response and growth of the mesophilic
Clostridium botulinum
ATCC 3502 at 15°C was demonstrated by induced expression of the TCS genes upon cold shock and impaired growth of the TCS mutants at 15°C.
Biodiesel Production by the Green Microalga Scenedesmus obliquus in a Recirculatory Aquaculture System
Authors: Mandal Shovon , Mallick Nirupama
Published: June 2012

Biodiesel production was examined with
Scenedesmus obliquus
in a recirculatory aquaculture system with fish pond discharge and poultry litter to couple with waste treatment. Lipid productivity of 14,400 liter ha
−1
year
−1
was projected with 11 cultivation cycles per year. The fuel properties of the biodiesel produced adhered to Indian and international standards.
Forward Genetic
In Planta
Screen for Identification of Plant-Protective Traits of Sphingomonas sp. Strain Fr1 against Pseudomonas syringae DC3000
Authors: Vogel Christine , Innerebner Gerd , Zingg Judith , Guder Jan , Vorholt Julia A.
Published: June 2012

Sphingomonas
sp. strain Fr1 has recently been shown to protect
Arabidopsis thaliana
against the bacterial leaf pathogen
Pseudomonas syringae
DC3000. Here, we describe a forward genetic
in planta
screen to identify genes in
Sphingomonas
sp. Fr1 necessary for this effect. About 5,000
Sphingomonas
sp. Fr1 mini-Tn
5
mutants were assayed for a defect in plant protection against a
luxCDABE
-tagged
P. syringae
DC3000 derivative in a space-saving 24-well plate system. The bioluminescence of the pathogen was used as the indicator of pathogen proliferation and allowed for the identification of
Sphingomonas
sp. Fr1 mutants that had lost the ability to restrict pathogen growth before disease symptoms were visible. Potential candidates were validated using the same miniaturized experimental system. Of these mutants, 10 were confirmed as plant protection defective yet colonization competent. The mutants were subsequently evaluated in a previously described standard microbox system, and plants showed enhanced disease phenotypes after pathogen infection relative to those inoculated with the parental strain as a control. However, the disease severities were lower than those observed for control plants that were grown axenically prior to pathogen challenge, which suggests that several traits may contribute to plant protection. Transposon insertion sites of validated mutants with defects in plant protection were determined and mapped to 7 distinct genomic regions. In conclusion, the established screening protocol allowed us to identify mutations that affect plant protection, and it opens the possibility to uncover traits important for
in planta
microbe-microbe interactions.
Pathogenic Escherichia coli Found in Sewage Treatment Plants and Environmental Waters
Authors: Anastasi E. M. , Matthews B. , Stratton H. M. , Katouli M.
Published: June 2012

We previously demonstrated that some
Escherichia coli
strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264
E. coli
isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93
E. coli
strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic
E. coli
(IPEC) or uropathogenic
E. coli
(UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2
2
, B2
3
, D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental
E. coli
in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.
Development of a Direct Isolation Procedure for Free-Living Diazotrophs under Controlled Hypoxic Conditions
Authors: Mirza Babur S. , Rodrigues Jorge L. M.
Published: June 2012

Free-living diazotrophs are diverse and ubiquitous in soil, contributing the nitrogen pool in natural ecosystems. The isolation of nitrogen-fixing microorganisms has relied on semisolid nitrogen-free medium enrichment, followed by multiple subculturing steps. These procedures limit the diversity of recovered isolates. In the current study, we investigated three different isolation strategies for free-living diazotrophs using a soil sample from the Amazon forest. The methods were (i) direct plating on solid nitrogen-free medium under a 2% O
2
concentration, (ii) enrichment in semisolid nitrogen-free medium before plating on solid nitrogen-free medium under 2% O
2
, and (iii) enrichment followed by subculturing in the semisolid nitrogen-free medium before plating on nitrogen containing medium under a 21% O
2
concentration. A total of 794 isolates were differentiated by their genomic fingerprinting patterns, and strains with unique profiles were identified on the basis of sequencing of their 16S rRNA gene. Isolates belonged to four bacterial phyla:
Proteobacteria
,
Firmicutes
,
Actinobacteria
, and
Bacteriodetes
. The novel strategy of combining a solid N-free medium and hypoxic conditions showed an increase of 62.6% in the diversity of diazotrophs in comparison to that obtained by the conventional semisolid medium-based methods. All isolates grew on the nitrogen-free medium under a 2% O
2
concentration, 78% of them showed the presence of the
nifH
gene, and 39% tested positive for acetylene reduction activity. Our results suggest that direct plating of soil dilutions on nitrogen-free solid medium under a 2% O
2
concentration is a useful strategy for the isolation of the diverse diazotrophic communities.
Effects of Sequential and Simultaneous Applications of Bacteriophages on Populations of Pseudomonas aeruginosa
In Vitro
and in Wax Moth Larvae
Authors: Hall Alex R. , De Vos Daniel , Friman Ville-Petri , Pirnay Jean-Paul , Buckling Angus
Published: June 2012

Interest in using bacteriophages to treat bacterial infections (phage therapy) is growing, but there have been few experiments comparing the effects of different treatment strategies on both bacterial densities and resistance evolution. While it is established that multiphage therapy is typically more effective than the application of a single phage type, it is not clear if it is best to apply phages simultaneously or sequentially. We tried single- and multiphage therapy against
Pseudomonas aeruginosa
PAO1
in vitro
, using different combinations of phages either simultaneously or sequentially. Across different phage combinations, simultaneous application was consistently equal or superior to sequential application in terms of reducing bacterial population density, and there was no difference (on average) in terms of minimizing resistance. Phage-resistant bacteria emerged in all experimental treatments and incurred significant fitness costs, expressed as reduced growth rate in the absence of phages. Finally, phage therapy increased the life span of wax moth larvae infected with
P. aeruginosa
, and a phage cocktail was the most effective short-term treatment. When the ratio of phages to bacteria was very high, phage cocktails cured otherwise lethal infections. These results suggest that while adding all available phages simultaneously tends to be the most successful short-term strategy, there are sequential strategies that are equally effective and potentially better over longer time scales.
Exploiting a Natural Auxotrophy for Genetic Selection
Authors: Ramage Elizabeth , Gallagher Larry , Manoil Colin
Published: June 2012

We exploited the natural histidine auxotrophy of
Francisella
species to develop
hisD
(encodes histidinol dehydrogenase) as a positive selection marker. A shuttle plasmid (pBR103) carrying
Escherichia coli hisD
and designed for cloning of PCR fragments replicated in both attenuated and highly virulent
Francisella
strains. During this work, we formulated a simplified defined growth medium for
Francisella novicida
.
Actinobacterial Nitrate Reducers and Proteobacterial Denitrifiers Are Abundant in N
2
O-Metabolizing Palsa Peat
Authors: Palmer Katharina , Horn Marcus A.
Published: June 2012

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N
2
O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland.
In situ
N
2
O fluxes were spatially variable, ranging from 0.01 to −0.02 μmol of N
2
O m
−2
h
−1
. Fertilization with nitrate stimulated
in situ
N
2
O emissions and N
2
O production in anoxic microcosms without apparent delay. N
2
O was subsequently consumed in microcosms. Maximal reaction velocities (
v
max
) of nitrate-dependent denitrification approximated 3 and 1 nmol of N
2
O per h per gram (dry weight [g
DW
]) in soil from 0 to 20 cm and below 20 cm of depth, respectively.
v
max
values of nitrite-dependent denitrification were 2- to 5-fold higher than the
v
max
nitrate-dependent denitrification, and
v
max
of N
2
O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N
2
O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of
narG
,
nirK
and
nirS
, and
nosZ
were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as
Actino
-,
Alpha
-, and
Betaproteobacteria
. Copy numbers of
nirS
always outnumbered those of
nirK
by 2 orders of magnitude. Copy numbers of
nirS
tended to be higher, while copy numbers of
narG
and
nosZ
tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N
2
O are associated with denitrification, (ii) actinobacterial nitrate reducers and
nirS
-type and
nosZ
-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N
2
O fluxes.
A Synthetic Arabinose-Inducible Promoter Confers High Levels of Recombinant Protein Expression in Hyperthermophilic Archaeon Sulfolobus islandicus
Authors: Peng Nan , Deng Ling , Mei Yuxia , Jiang Dongqing , Hu Yongmei , Awayez Mariana , Liang Yunxiang , She Qunxin
Published: June 2012

Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression in
Sulfolobus islandicus
, a hyperthermophilic crenarchaeon. Two expression vectors, pSeSD and pEXA, harboring 11 unique restriction sites were constructed. They contain coding sequences of two hexahistidine (6×His) peptide tags and those coding for two protease sites, the latter of which make it possible to remove the peptide tags from expressed recombinant proteins. While pEXA employed an
araS
promoter for protein expression, pSeSD utilized P

araS
-SD

, an
araS
derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P

araS
-SD

directed high levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-
lacS
lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started at multiple sites downstream of the 6×His codons. Intriguingly, inserting an RBS site upstream of the ATG codon regained the expression of the 6×His tag, as shown with pSeSD-N-
lacS
. These results have yielded novel insight into the archaeal translation mechanism. The crenarchaeon
Sulfolobus
can utilize N-terminal coding sequences of proteins to specify translation initiation in the absence of an RBS site.
Association of Campylobacter jejuni Metabolic Traits with Multilocus Sequence Types
Authors: de Haan Caroline P. A. , Llarena Ann-Katrin , Revez Joana , Hänninen Marja-Liisa
Published: June 2012

In this study, we describe the association of three
Campylobacter jejuni
metabolism-related traits, γ-glutamyl-transpeptidase (GGT), fucose permease (
fucP
), and secreted
l
-asparaginase [
ansB
(s)], with multilocus sequence types (STs). A total of 710
C. jejuni
isolates with known STs were selected and originated from humans, poultry, bovines, and the environment. Among these isolates, we found 31.1% to produce GGT and 49.3% and 30.3% to be positive for
ansB
(s) and
fucP
, respectively. The combination of GGT production, the presence of
ansB
(s), and the absence of
fucP
was associated with ST-22, ST-586, and the ST-45 and ST-283 clonal complexes (CCs), which were the main STs and CCs found among the human and chicken isolates. The ST-21 CC was associated with the presence of
fucP
and was the major CC among the bovine isolates. Although the ST-61 CC was the second major CC among the bovine isolates, these isolates did not have any of the markers studied, making the role of
fucP
in bovine gut colonization questionable. The ST-45 CC was subdivided into three groups that were attributed solely to ST-45. One group showed a marker combination described previously, another group was found to be positive for
ansB
(s) only, and the third group did not have any of the markers studied. These results suggest that the host association of these markers seems to be indirect and may arise as a consequence of host-ST and -CC associations. Thus, a representative collection of STs should be tested to draw sensible conclusions in similar studies.
The Novel Kasugamycin 2′-
N
-Acetyltransferase Gene
aac(2′)-IIa
, Carried by the IncP Island, Confers Kasugamycin Resistance to Rice-Pathogenic Bacteria
Authors: Yoshii Atsushi , Moriyama Hiromitsu , Fukuhara Toshiyuki
Published: June 2012

Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus
Magnaporthe grisea
but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by
Burkholderia glumae
or
Acidovorax avenae
subsp.
avenae
, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant
B. glumae
and
A. avenae
isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene,
aac(2′)-IIa
, encoding a KSM 2′-
N
-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2′)-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The
aac(2′)-IIa
gene from
B. glumae
strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the
aac(2′)-IIa
gene were detected. These results indicate that the
aac(2′)-IIa
gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the
aac(2′)-IIa
gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM.
Transovarial Transmission of Rickettsia spp. and Organ-Specific Infection of the Whitefly Bemisia tabaci
Authors: Brumin Marina , Levy Maggie , Ghanim Murad
Published: June 2012

The whiteflyBemisia tabaciis a cosmopolitan insect pest that harborsPortiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts inB. tabaciare generally associated with the bacteriome, ensuring their vertical transmission; however,Rickettsiais an exception and occupies most of the body cavity, except the bacteriome. The mode ofRickettsiatransfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show thatRickettsiainfects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome.Rickettsiainvades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations.Rickettsiawas localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected byRickettsiaincluded the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association betweenRickettsiaand theB. tabacidigestive system might be important for digestive purposes. The vertical transmission ofRickettsiato subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.
Two Gene Clusters Coordinate Galactose and Lactose Metabolism in Streptococcus gordonii
Authors: Zeng Lin , Martino Nicole C. , Burne Robert A.
Published: June 2012

Streptococcus gordonii
is an early colonizer of the human oral cavity and an abundant constituent of oral biofilms. Two tandemly arranged gene clusters, designated
lac
and
gal
, were identified in the
S. gordonii
DL1 genome, which encode genes of the tagatose pathway (
lacABCD
) and sugar phosphotransferase system (PTS) enzyme II permeases. Genes encoding a predicted phospho-β-galactosidase (LacG), a DeoR family transcriptional regulator (LacR), and a transcriptional antiterminator (LacT) were also present in the clusters. Growth and PTS assays supported that the permease designated EII
Lac
transports lactose and galactose, whereas EII
Gal
transports galactose. The expression of the gene for EII
Gal
was markedly upregulated in cells growing on galactose. Using promoter-
cat
fusions, a role for LacR in the regulation of the expressions of both gene clusters was demonstrated, and the
gal
cluster was also shown to be sensitive to repression by CcpA. The deletion of
lacT
caused an inability to grow on lactose, apparently because of its role in the regulation of the expression of the genes for EII
Lac
, but had little effect on galactose utilization.
S. gordonii
maintained a selective advantage over
Streptococcus mutans
in a mixed-species competition assay, associated with its possession of a high-affinity galactose PTS, although
S. mutans
could persist better at low pHs. Collectively, these results support the concept that the galactose and lactose systems of
S. gordonii
are subject to complex regulation and that a high-affinity galactose PTS may be advantageous when
S. gordonii
is competing against the caries pathogen
S. mutans
in oral biofilms.
Arcobacter in Lake Erie Beach Waters: an Emerging Gastrointestinal Pathogen Linked with Human-Associated Fecal Contamination
Authors: Lee Cheonghoon , Agidi Senyo , Marion Jason W. , Lee Jiyoung
Published: June 2012

The genus
Arcobacter
has been associated with human illness and fecal contamination by humans and animals. To better characterize the health risk posed by this emerging waterborne pathogen, we investigated the occurrence of
Arcobacter
spp. in Lake Erie beach waters. During the summer of 2010, water samples were collected 35 times from the Euclid, Villa Angela, and Headlands (East and West) beaches, located along Ohio's Lake Erie coast. After sample concentration,
Arcobacter
was quantified by real-time PCR targeting the
Arcobacter
23S rRNA gene. Other fecal genetic markers (
Bacteroides
16S rRNA gene [HuBac],
Escherichia coli uidA
gene,
Enterococcus
23S rRNA gene, and tetracycline resistance genes) were also assessed.
Arcobacter
was detected frequently at all beaches, and both the occurrence and densities of
Arcobacter
spp. were higher at the Euclid and Villa Angela beaches (with higher levels of fecal contamination) than at the East and West Headlands beaches. The
Arcobacter
density in Lake Erie beach water was significantly correlated with the human-specific fecal marker HuBac according to Spearman's correlation analysis (
r
= 0.592;
P
< 0.001). Phylogenetic analysis demonstrated that most of the identified
Arcobacter
sequences were closely related to
Arcobacter cryaerophilus
, which is known to cause gastrointestinal diseases in humans. Since human-pathogenic
Arcobacter
spp. are linked to human-associated fecal sources, it is important to identify and manage the human-associated contamination sources for the prevention of
Arcobacter
-associated public health risks at Lake Erie beaches.
Solubility-Promoting Function of Hsp90 Contributes to Client Maturation and Robust Cell Growth
Authors: Pursell Natalie W. , Mishra Parul , Bolon Daniel N. A.
Published: June 2012

The Hsp90 chaperone is required for the maturation of signal transduction clients, including many kinases and nuclear steroid hormone receptors. The binding and hydrolysis of ATP by Hsp90 drive conformational rearrangements in three structure domains. Two intrinsically disordered regions of Hsp90 located between these domains and at the C terminus have traditionally been considered to impart flexibility. We discovered that the charged nature of these acid-rich disordered regions imparts a solubility-promoting function to Hsp90 that is important for its cellular activity in yeast. Both the solubility-promoting function and ATPase activity must occur in the same Hsp90 molecule in order to support robust growth, suggesting that the solubility-promoting function is required during the ATP-driven client maturation process. Expression of model clients together with Hsp90 variants indicated interdependent solubilities mediated by the aggregation propensities of both the client and Hsp90. We propose a model whereby the charge-rich disordered regions of Hsp90 serve a solubility-promoting function important for complexes with aggregation-prone clients. These findings demonstrate a novel biological function of the intrinsically disordered regions in Hsp90 and provide a compelling rationale for why their charged properties are conserved throughout eukaryotic evolution.
Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae
Authors: Jungbluth Marc , Mösch Hans-Ulrich , Taxis Christof
Published: June 2012

In
Saccharomyces cerevisiae
, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (
ady2
Δ) or carbonic anhydrase (
nce103
Δ) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.
SrfJ, a Salmonella Type III Secretion System Effector Regulated by PhoP, RcsB, and IolR
Authors: Cordero-Alba Mar , Bernal-Bayard Joaquín , Ramos-Morales Francisco
Published: June 2012

Virulence-related type III secretion systems are present in many Gram-negative bacterial pathogens. These complex devices translocate proteins, called effectors, from the bacterium into the eukaryotic host cell. Here, we identify the product of
srfJ
, a
Salmonella enterica
serovar Typhimurium gene regulated by SsrB, as a new substrate of the type III secretion system encoded by
Salmonella
pathogenicity island 2. The N-terminal 20-amino-acid segment of SrfJ was recognized as a functional secretion and translocation signal specific for this system. Transcription of
srfJ
was positively regulated by the PhoP/PhoQ system in an SsrB-dependent manner and was negatively regulated by the Rcs system in an SsrB-independent manner. A screen for regulators of an
srfJ-lacZ
transcriptional fusion using the T-POP transposon identified IolR, the regulator of genes involved in
myo
-inositol utilization, as an
srfJ
repressor. Our results suggest that SrfJ is synthesized both inside the host, in response to intracellular conditions, and outside the host, in
myo
-inositol-rich environments.
Growing Unculturable Bacteria
Authors: Stewart Eric J.
Published: June 2012

The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field.
YqjD Is an Inner Membrane Protein Associated with Stationary-Phase Ribosomes in Escherichia coli
Authors: Yoshida Hideji , Maki Yasushi , Furuike Shou , Sakai Akiko , Ueta Masami , Wada Akira
Published: June 2012

Here, we provide evidence that YqjD, a hypothetical protein of
Escherichia coli
, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region. Interestingly,
E. coli
possesses two paralogous proteins of YqjD, ElaB and YgaM, which are expressed and bind to ribosomes in a similar manner to YqjD. Overexpression of YqjD leads to inhibition of cell growth. It has been suggested that YqjD loses ribosomal activity and localizes ribosomes to the membrane during the stationary phase.
Activator Role of the Pneumococcal Mga-Like Virulence Transcriptional Regulator
Authors: Solano-Collado Virtu , Espinosa Manuel , Bravo Alicia
Published: June 2012

Global transcriptional regulators that respond to specific environmental signals are crucial in bacterial pathogenesis. In the case of the Gram-positive pathogen
Streptococcus pneumoniae
(the pneumococcus), the
sp1800
gene of the clinical isolate TIGR4 encodes a protein that exhibits homology to the Mga “stand-alone” response regulator of the group A
Streptococcus
. Such a pneumococcal protein was shown to play a significant role in both nasopharyngeal colonization and development of pneumonia in murine infection models. Moreover, it was shown to repress the expression of several genes located within the
rlrA
pathogenicity islet. The pneumococcal R6 strain, which derives from the D39 clinical isolate, lacks the
rlrA
islet but has a gene (here named

mga
Spn

) equivalent to the
sp1800
gene. In this work, and using
in vivo
approaches, we have identified the promoter of the

mga
Spn

gene (
Pmga
) and demonstrated that four neighboring open reading frames of unknown function (
spr1623
to
spr1626
) constitute an operon. Transcription of this operon is under the control of two promoters (
P1623A
and
P1623B
) that are divergent from the
Pmga
promoter. Furthermore, we have shown that the Mga

Spn

protein activates the
P1623B
promoter
in vivo
. This activation requires sequences located around 50 to 120 nucleotides upstream of the
P1623B
transcription start site. By DNase I footprinting assays, we have also demonstrated that such a region includes an Mga

Spn

binding site. This is the first report on the activator role of the pneumococcal Mga-like protein.
Mycobacterium tuberculosis Protein Kinase K Enables Growth Adaptation through Translation Control
Authors: Malhotra Vandana , Okon Blessing P. , Clark-Curtiss Josephine E.
Published: June 2012

Mycobacterium tuberculosis
serine/threonine protein kinases (STPKs) are responsible for orchestrating critical metabolic and physiological changes that dictate mycobacterial growth adaptation. Previously, we established that PknK participates in regulatory pathways that slow the growth of
M. tuberculosis
in a variety of
in vitro
stress environments and during persistent infection in mice. In the present study, we have elaborated on the mechanism of PknK-mediated regulation. Through transcription profiling of wild-type H37Rv and a Δ
pknK
mutant strain during logarithmic and stationary growth phases, we determined that PknK regulates the expression of a large subset of tRNA genes so that regulation is synchronized with growth phase and cellular energy status. Elevated levels of wild-type
M. tuberculosis
PknK (PknK

Mtb

), but not phosphorylation-defective PknK

Mtb

, in
Mycobacterium smegmatis
cause significant retardation of the growth rate and altered colony morphology. We investigated a role for PknK in translational control and established that PknK directs the inhibition of
in vitro
transcription and translation processes in a phosphorylation-dependent manner. Increasing concentrations of ATP or PknK exert cooperative effects and enhance the inhibitory function of PknK. Furthermore, truncation and mutational analyses of PknK revealed that PknK is autoregulated via intramolecular interactions with its C-terminal region. Significantly, the invariant lysine 55 residue was only essential for activity in the full-length PknK protein, and the truncated mutant proteins were active. A model for PknK autoregulation is proposed and discussed.