bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermal and Nonthermal Effects of Discontinuous Microwave Exposure (2.45 Gigahertz) on the Cell Membrane of Escherichia coli
Authors: Rougier Carole , Prorot Audrey , Chazal Philippe , Leveque Philippe , Leprat Patrick 
Published: June 2014

ABSTRACT

The aim of this study was to investigate the effects on the cell membranes of
Escherichia coli
of 2.45-GHz microwave (MW) treatment under various conditions with an average temperature of the cell suspension maintained at 37°C in order to examine the possible thermal versus nonthermal effects of short-duration MW exposure. To this purpose, microwave irradiation of bacteria was performed under carefully defined and controlled parameters, resulting in a discontinuous MW exposure in order to maintain the average temperature of the bacterial cell suspensions at 37°C.
Escherichia coli
cells were exposed to 200- to 2,000-W discontinuous microwave (DW) treatments for different periods of time. For each experiment, conventional heating (CH) in a water bath at 37°C was performed as a control. The effects of DW exposure on cell membranes was investigated using flow cytometry (FCM), after propidium iodide (PI) staining of cells, in addition to the assessment of intracellular protein release in bacterial suspensions. No effect was detected when bacteria were exposed to conventional heating or 200 W, whereas cell membrane integrity was slightly altered when cell suspensions were subjected to powers ranging from 400 to 2,000 W. Thermal characterization suggested that the temperature reached by the microwave-exposed samples for the contact time studied was not high enough to explain the measured modifications of cell membrane integrity. Because the results indicated that the cell response is power dependent, the hypothesis of a specific electromagnetic threshold effect, probably related to the temperature increase, can be advanced.
A Third Subunit in Ancestral Cytochrome
c
-Dependent Nitric Oxide Reductases
Authors: Bricio C. , Alvarez L. , San Martin M. , Schurig-Briccio L. A. , Gennis R. B. , Berenguer J. 
Published: June 2014

ABSTRACT

Reduction of NO to N
2
O by denitrifiying bacteria is catalyzed either by a monomeric quinol-nitric oxide reductase (qNor) or by a heterodimeric cytochrome
c
-dependent nitric oxide reductase (cNor). In ancient thermophilic bacteria belonging to the
Thermales
and
Aquificales
phylogenetic groups, the cluster encoding the cNor includes a small third gene (
norH
), in addition to those encoding homologues to the subunits of a typical cNor (
norC
and
norB
). We show in
Thermus thermophilus
that the three genes are cotranscribed in a single mRNA from an inducible promoter. The isolation of individual
nor
mutants and the production
in vivo
of His-tagged NorH protein followed by immobilized-metal affinity chromatography (IMAC) allowed us to conclude that NorH constitutes a third subunit of the cNor from
T. thermophilus
, which is involved in denitrification
in vivo
, likely allowing more efficient electron transport to cNor.
Effect of the Surfactant Tween 80 on the Detachment and Dispersal of Salmonella enterica Serovar Thompson Single Cells and Aggregates from Cilantro Leaves as Revealed by Image Analysis
Authors: Brandl Maria T. , Huynh Steven 
Published: June 2014

ABSTRACT

Salmonella enterica
has the ability to form biofilms and large aggregates on produce surfaces, including on cilantro leaves. Aggregates of
S. enterica
serovar Thompson that remained attached to cilantro leaves after rigorous washing and that were present free or bound to dislodged leaf tissue in the wash suspension were observed by confocal microscopy. Measurement of
S.
Thompson population sizes in the leaf washes by plate counts failed to show an effect of 0.05% Tween 80 on the removal of the pathogen from cilantro leaves 2 and 6 days after inoculation. On the contrary, digital image analysis of micrographs of single cells and aggregates of green fluorescent protein (GFP)-
S.
Thompson present in cilantro leaf washes revealed that single cells represented 13.7% of the cell assemblages in leaf washes containing Tween 80, versus 9.3% in those without the surfactant. Moreover, Tween 80 decreased the percentage of the total
S.
Thompson cell population located in aggregates equal to or larger than 64 cells from 9.8% to 4.4% (
P
< 0.05). Regression analysis of the frequency distribution of aggregate size in leaf washes with and without Tween 80 showed that the surfactant promoted the dispersal of cells from large aggregates into smaller ones and into single cells (
P
< 0.05). Our study underlines the importance of investigating bacterial behavior at the scale of single cells in order to uncover trends undetectable at the population level by bacterial plate counts. Such an approach may provide valuable information to devise strategies aimed at enhancing the efficacy of produce sanitization treatments.
A Novel Immunofluorescence Flow Cytometry Technique Detects the Expansion of Brown Tides Caused by Aureoumbra lagunensis to the Caribbean Sea
Authors: Koch F. , Kang Y. , Villareal T. A. , Anderson D. M. , Gobler C. J. 
Published: June 2014

ABSTRACT

During the past 3 decades, brown tides caused by the pelagophytes
Aureococcus anophagefferens
and
Aureoumbra lagunensis
have caused ecological and economic damage to coastal ecosystems across the globe. While blooms of
A. lagunensis
had previously been confined to Texas, in 2012, an expansive brown tide occurred on Florida's East Coast, causing widespread disruption within the Indian River and Mosquito Lagoons and generating renewed interest in this organism. A major impediment to detailed investigations of
A. lagunensis
in an ecosystem setting has been the absence of a rapid and reliable method for cell quantification. The combination of their small size (3 to 5 μm) and nondescript extracellular features makes identification and enumeration of these cells with conventional methods a challenge. Here we report the development of an immunological-based flow cytometry method that uses a fluorescently labeled antibody developed against
A. lagunensis
. This method is species specific, sensitive (detection limit of 1.5 × 10
3
cells ml
−1
), precise (1% relative standard deviation of replicated samples), and accurate (108% ± 8% recovery of spiked samples) over a wide range of cell concentrations. Furthermore, this method effectively quantifies
A. lagunensis
in both glutaraldehyde- and formalin-preserved samples, yields a high throughput of samples (∼35 samples h
−1
), and is cost-effective, making it an ideal tool for managers and scientists. This method successfully documented the recurrence of a brown tide bloom in Florida in 2013. Bloom densities were highest in June (>2.0 × 10
6
cells ml
−1
) and spanned >60 km from the Ponce de Leon inlet in the northern Mosquito Lagoon south to Titusville in the Indian River Lagoon. Low levels of
A. lagunensis
cells were found >250 km south of this region. This method also quickly and accurately identified
A. lagunensis
as the causative agent of a 2013 brown tide bloom in Guantanamo Bay, Cuba, and thus should prove useful for both quantifying the dynamics of ongoing blooms of
A. lagunensis
as well as documenting new outbreaks of this harmful alga.
Antifouling Coatings Influence both Abundance and Community Structure of Colonizing Biofilms: a Case Study in the Northwestern Mediterranean Sea
Authors: Camps Mercedes , Barani Aude , Gregori Gérald , Bouchez Agnès , Le Berre Brigitte , Bressy Christine , Blache Yves , Briand Jean-François 
Published: June 2014

ABSTRACTWhen immersed in seawater, substrates are rapidly colonized by both micro- and macroorganisms. This process is responsible for important economic and ecological prejudices, particularly when related to ship hulls or aquaculture nets. Commercial antifouling coatings are supposed to reduce biofouling, i.e., micro- and macrofoulers. In this study, biofilms that primarily settled on seven different coatings (polyvinyl chloride [PVC], a fouling release coating [FRC], and five self-polishing copolymer coatings [SPC], including four commercial ones) were quantitatively studied, after 1 month of immersion in summer in the Toulon Bay (Northwestern Mediterranean Sea, France), by using flow cytometry (FCM), microscopy, and denaturing gradient gel electrophoresis. FCM was used after a pretreatment to separate cells from the biofilm matrix, in order to determine densities of heterotrophic bacteria, picocyanobacteria, and pico- and nanoeukaryotes on these coatings. Among diatoms, the only microphytobenthic class identified by microscopy,Licmophora,Navicula, andNitzschiawere determined to be the dominant taxa. Overall, biocide-free coatings showed higher densities than all other coatings, except for one biocidal coating, whatever the group of microorganisms. Heterotrophic bacteria always showed the highest densities, and diatoms showed the lowest, but the relative abundances of these groups varied depending on the coating. In particular, the copper-free SPC failed to prevent diatom settlement, whereas the pyrithione-free SPC exhibited high picocyanobacterial density. These results highlight the interest in FCM for antifouling coating assessment as well as specific selection among microbial communities by antifouling coatings.
Vertebrate Decomposition Is Accelerated by Soil Microbes
Authors: Lauber Christian L. , Metcalf Jessica L. , Keepers Kyle , Ackermann Gail , Carter David O. , Knight Rob 
Published: June 2014

ABSTRACT
Carrion decomposition is an ecologically important natural phenomenon influenced by a complex set of factors, including temperature, moisture, and the activity of microorganisms, invertebrates, and scavengers. The role of soil microbes as decomposers in this process is essential but not well understood and represents a knowledge gap in carrion ecology. To better define the role and sources of microbes in carrion decomposition, lab-reared mice were decomposed on either (i) soil with an intact microbial community or (ii) soil that was sterilized. We characterized the microbial community (16S rRNA gene for bacteria and archaea, and the 18S rRNA gene for fungi and microbial eukaryotes) for three body sites along with the underlying soil (i.e., gravesoils) at time intervals coinciding with visible changes in carrion morphology. Our results indicate that mice placed on soil with intact microbial communities reach advanced stages of decomposition 2 to 3 times faster than those placed on sterile soil. Microbial communities associated with skin and gravesoils of carrion in stages of active and advanced decay were significantly different between soil types (sterile versus untreated), suggesting that substrates on which carrion decompose may partially determine the microbial decomposer community. However, the source of the decomposer community (soil- versus carcass-associated microbes) was not clear in our data set, suggesting that greater sequencing depth needs to be employed to identify the origin of the decomposer communities in carrion decomposition. Overall, our data show that soil microbial communities have a significant impact on the rate at which carrion decomposes and have important implications for understanding carrion ecology.
Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack
Authors: Bakshi Somenath , Choi Heejun , Rangarajan Nambirajan , Barns Kenneth J. , Bratton Benjamin P. , Weisshaar James C. 
Published: June 2014

ABSTRACT

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live
Escherichia coli
, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative
E. coli
and the Gram-positive
Bacillus subtilis
. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the
E. coli
cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.
Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose
Authors: Currie D. H. , Guss A. M. , Herring C. D. , Giannone R. J. , Johnson C. M. , Lankford P. K. , Brown S. D. , Hettich R. L. , Lynd L. R. 
Published: June 2014

ABSTRACT

Thermoanaerobacterium saccharolyticum
, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by
T. saccharolyticum
for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (
Tsac_1445
through
Tsac_1464
for xylan/xylose and
Tsac_1344
through
Tsac_1349
for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in
T. saccharolyticum
, and deletion of the
Tsac_0361
gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C
5
sugars.
Distribution of Virulence-Associated Genes and Genetic Relationships in Non-O1/O139 Vibrio cholerae Aquatic Isolates from China
Authors: Li Fengjuan , Du Pengcheng , Li Baisheng , Ke Changwen , Chen Aiping , Chen Jie , Zhou Haijian , Li Jie , Morris J. Glenn , Kan Biao , Wang Duochun 
Published: June 2014

ABSTRACT

Non-O1/O139
Vibrio cholerae
is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of
V. cholerae
non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for
ctxB
and harbored a rare genotype; 10 (3.4%) isolates carried several types of
rstR
sequences, eight of which carried rare types of toxin-coregulated pili (
tcpA
). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including
mshA
(95.6%),
hlyA
(95.3%),
rtxC
(89.8%),
rtxA
(82.7%), IS1004 (52.9%),
chxA
(30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (
n
= 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The
V. cholerae
non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.
Extracellular Enzyme Activity and Microbial Diversity Measured on Seafloor Exposed Basalts from Loihi Seamount Indicate the Importance of Basalts to Global Biogeochemical Cycling
Authors: Jacobson Meyers Myrna E. , Sylvan Jason B. , Edwards Katrina J. 
Published: June 2014

ABSTRACT

Seafloor basalts are widely distributed and host diverse prokaryotic communities, but no data exist concerning the metabolic rates of the resident microbial communities. We present here potential extracellular enzyme activities of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) measured on basalt samples from different locations on Loihi Seamount, HI, coupled with analysis of prokaryotic biomass and pyrosequencing of the bacterial 16S rRNA gene. The community maximum potential enzyme activity (
V
max
) of LAP ranged from 0.47 to 0.90 nmol (g rock)
−1
h
−1
; the
V
max
for AP was 28 to 60 nmol (g rock)
−1
h
−1
. The

K
m

of LAP ranged from 26 to 33 μM, while the

K
m

for AP was 2 to 7 μM. Bacterial communities on Loihi basalts were comprised primarily of
Alpha
-,
Delta
-, and
Gammaproteobacteria
,
Bacteroidetes
, and
Planctomycetes
. The putative ability to produce LAP is evenly distributed across the most commonly detected bacterial orders, but the ability to produce AP is likely dominated by bacteria in the orders
Xanthomonadales
,
Flavobacteriales
, and
Planctomycetales
. The enzyme activities on Loihi basalts were compared to those of other marine environments that have been studied and were found to be similar in magnitude to those from continental shelf sediments and orders of magnitude higher than any measured in the water column, demonstrating that the potential for exposed basalts to transform organic matter is substantial. We propose that microbial communities on basaltic rock play a significant, quantifiable role in benthic biogeochemical processes.
To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB
Authors: Sznajder Anna , Jendrossek Dieter 
Published: June 2014

ABSTRACT

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of
Ralstonia eutropha
H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules
in vitro
. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from Δ
phaP1
or Δ
phaP1-phaP5
mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions
in vivo
. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of
phaZd1
,
phaZd2
, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor
lacZ
fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true
in vivo
functions of PhaZd1 and PhaZd2 remain obscure.
Revision of N2O-Producing Pathways in the Ammonia-Oxidizing Bacterium Nitrosomonas europaea ATCC 19718
Authors: Kozlowski Jessica A. , Price Jennifer , Stein Lisa Y. 
Published: June 2014

ABSTRACTNitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N2O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N2O production by ammonia oxidizers. The growth phenotypes of theNitrosomonas europaeaATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O2tensions. Anoxic resting-cell assays and instantaneous nitrite (NO2−) reduction experiments were done to assess the ability of the wild-type and mutantN. europaeastrains to produce N2O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation ofN. europaeaand showed that NorB is involved in N2O production during growth at both atmospheric and reduced O2tensions. Anoxic resting-cell assays and measurements of instantaneous NO2−reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N2O production byN. europaeaand will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.
Use of Silica-Encapsulated Pseudomonas sp. Strain NCIB 9816-4 in Biodegradation of Novel Hydrocarbon Ring Structures Found in Hydraulic Fracturing Waters
Authors: Aukema Kelly G. , Kasinkas Lisa , Aksan Alptekin , Wackett Lawrence P. 
Published: June 2014

ABSTRACT

The most problematic hydrocarbons in hydraulic fracturing (fracking) wastewaters consist of fused, isolated, bridged, and spiro ring systems, and ring systems have been poorly studied with respect to biodegradation, prompting the testing here of six major ring structural subclasses using a well-characterized bacterium and a silica encapsulation system previously shown to enhance biodegradation. The direct biological oxygenation of spiro ring compounds was demonstrated here. These and other hydrocarbon ring compounds have previously been shown to be present in flow-back waters and waters produced from hydraulic fracturing operations.
Pseudomonas
sp. strain NCIB 9816-4, containing naphthalene dioxygenase, was selected for its broad substrate specificity, and it was demonstrated here to oxidize fundamental ring structures that are common in shale-derived waters but not previously investigated with this or related enzymes.
Pseudomonas
sp. NCIB 9816-4 was tested here in the presence of a silica encasement, a protocol that has previously been shown to protect bacteria against the extremes of salinity present in fracking wastewaters. These studies demonstrate the degradation of highly hydrophobic compounds by a silica-encapsulated model bacterium, demonstrate what it may not degrade, and contribute to knowledge of the full range of hydrocarbon ring compounds that can be oxidized using
Pseudomonas
sp. NCIB 9816-4.