bluuebunny/crossref_metadata_2025_split huggingface dataset:
Real-Time Microsensor Measurement of Local Metabolic Activities in
Ex Vivo
Dental Biofilms Exposed to Sucrose and Treated with Chlorhexidine
Authors: von Ohle Christiane , Gieseke Armin , Nistico Laura , Decker Eva Maria , deBeer Dirk , Stoodley Paul 
Published: January 2010

ABSTRACT

Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms
ex vivo
by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces
in vivo
. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 μm) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.
Sequence Analysis of
Leuconostoc mesenteroides
Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation
Authors: Lu Z. , Altermann E. , Breidt F. , Kozyavkin S. 
Published: January 2010

ABSTRACT

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB).
Leuconostoc mesenteroides
initiates fermentation. As fermentation proceeds,
L. mesenteroides
dies off and other LAB complete the fermentation. Phages infecting
L. mesenteroides
may significantly influence the die-off of
L. mesenteroides
. However, no
L. mesenteroides
phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of
L. mesenteroides
phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules.
In silico
analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several
Lactococcus
phages. To our knowledge, Φ1-A4 is the first genetically characterized
L. mesenteroides
phage.
Soil Resources Influence Spatial Patterns of Denitrifying Communities at Scales Compatible with Land Management
Authors: Enwall Karin , Throbäck Ingela N. , Stenberg Maria , Söderström Mats , Hallin Sara 
Published: January 2010

ABSTRACTKnowing spatial patterns of functional microbial guilds can increase our understanding of the relationships between microbial community ecology and ecosystem functions. Using geostatistical modeling to map spatial patterns, we explored the distribution of the community structure, size, and activity of one functional group in N cycling, the denitrifiers, in relation to 23 soil parameters over a 44-ha farm divided into one organic and one integrated crop production system. The denitrifiers were targeted by thenirSandnirKgenes that encode the two mutually exclusive types of nitrite reductases, the cd1heme-type and copper reductases, respectively. The spatial pattern of the denitrification activity genes was reflected by the maps of the abundances ofnirgenes. For the community structure, only the maps of thenirScommunity were related to the activity. The activity was correlated with nitrate and dissolved organic nitrogen and carbon, whereas the gene pools for denitrification, in terms of size and composition, were influenced by the soil structure. For thenirScommunity, pH and soil nutrients were also important in shaping the community. The only unique parameter related to thenirKcommunity was the soil Cu content. However, the spatial pattern of thenirKdenitrifiers corresponded to the division of the farm into the two cropping systems. The different community patterns, together with the spatial distribution of thenirS/nirKabundance ratio, suggest habitat selection on thenirS- andnirK-type denitrifiers. Our findings constitute a first step in identifying niches for denitrifiers at scales relevant to land management.
Functional Genomic Study of Exogenous
n
-Butanol Stress in
Escherichia coli
Authors: Rutherford Becky J. , Dahl Robert H. , Price Richard E. , Szmidt Heather L. , Benke Peter I. , Mukhopadhyay Aindrila , Keasling Jay D. 
Published: January 2010

ABSTRACT

n
-Butanol has been proposed as an alternative biofuel to ethanol, and several industrially used microbes, including
Escherichia coli
, have been engineered to produce it. Unfortunately,
n
-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell-wide studies were conducted at the transcript, protein, and metabolite levels to obtain a global view of the
n
-butanol stress response. Analysis of the data indicates that
n
-butanol stress has components common to other stress responses, including perturbation of respiratory functions (
nuo
and
cyo
operons), oxidative stress (
sodA
,
sodC
, and
yqhD
), heat shock and cell envelope stress (
rpoE
,
clpB
,
htpG
,
cpxR
, and
cpxP
), and metabolite transport and biosynthesis (
malE
and
opp
operon). Assays using fluorescent dyes indicated a large increase in reactive oxygen species during
n
-butanol stress, confirming observations from the microarray and proteomics measurements. Mutant strains with mutations in several genes whose products changed most dramatically during
n
-butanol stress were examined for increased sensitivity to
n
-butanol. Results from these analyses allowed identification of key genes that were recruited to alleviate oxidative stress, protein misfolding, and other causes of growth defects. Cellular engineering based on these cues may assist in developing a high-titer,
n
-butanol-producing host.
Roles of Siderophores, Oxalate, and Ascorbate in Mobilization of Iron from Hematite by the Aerobic Bacterium
Pseudomonas mendocina
Authors: Dehner Carolyn A. , Awaya Jonathan D. , Maurice Patricia A. , DuBois Jennifer L. 
Published: January 2010

ABSTRACT

In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (α-Fe
2
O
3
) by the obligate aerobe
Pseudomonas mendocina
ymp. A site-directed Δ
pmhA
mutant that was not capable of producing functional siderophores (i.e., siderophore
−
phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 μM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore
−
strain was ∼50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 μM) alone restored the growth of the siderophore
−
culture to the WT levels. A higher concentration of oxalate (100 μM) had little effect on the growth of a siderophore
−
culture; however, addition of 0.1 μM DFO-B and 100 μM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.
Molecular Indicators Used in the Development of Predictive Models for Microbial Source Tracking
Authors: Ballesté Elisenda , Bonjoch Xavier , Belanche Lluís A. , Blanch Anicet R. 
Published: January 2010

ABSTRACT

A number of chemical, microbial, and eukaryotic indicators have been proposed as indicators of fecal pollution sources in water bodies. No single one of the indicators tested to date has been able to determine the source of fecal pollution in water. However, the combined use of different indicators has been demonstrated to be the best way of defining predictive models suitable for determining fecal pollution sources. Molecular methods are promising tools that could complement standard microbiological water analysis. In this study, the feasibility of some proposed molecular indicators for microbial source tracking (MST) was compared (names of markers are in parentheses): host-specific
Bacteroidetes
(HF134, HF183, CF128, and CF193),
Bifidobacterium adolescentis
(ADO),
Bifidobacterium dentium
(DEN), the gene
esp
of
Enterococcus faecium
, and host-specific mitochondrial DNA associated with humans, cattle, and pigs (Humito, Bomito, and Pomito, respectively). None of the individual molecular markers tested enabled 100% source identification. They should be combined with other markers to raise sensitivity and specificity and increase the number of sources that are identified. MST predictive models using only these molecular markers were developed. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of identification of fecal sources. The combined use of three molecular markers (ADO, Bomito, and Pomito) enabled correct identification of 75.7% of the samples, with differentiation between human, swine, bovine, and poultry sources. Discrimination between human and nonhuman fecal pollution was possible using two markers: ADO and Pomito (84.6% correct identification). The percentage of correct identification increased with the number of markers analyzed. The best predictive model for distinguishing human from nonhuman fecal sources was based on 5 molecular markers (HF134, ADO, DEN, Bomito, and Pomito) and provided 90.1% correct classification.
Stress-Induced Hsp70 Gene Expression and Inactivation of
Cryptosporidium parvum
Oocysts by Chlorine-Based Oxidants
Authors: Bajszár George , Dekonenko Alexander 
Published: January 2010

ABSTRACT

Our research on the mechanisms of action of chlorine-based oxidants on
Cryptosporidium parvum
oocysts in water revealed a dual-phase effect: (i) response to oxidative stress, which was demonstrated by induced expression of the Hsp70 heat shock gene, and (ii) oocyst inactivation as a result of long-term exposure to oxidants. The relative biocidal effects of sodium hypochlorite (bleach) and electrolytically generated mixed oxidant solution (MOS) on
C. parvum
oocysts were compared at identical free chlorine concentrations. Oocyst inactivation was determined by quantitative reverse transcription-PCR (qRT-PCR) amplification of the heat-induced Hsp70 mRNA and compared with tissue culture infectivity. According to both assays, within the range between 25 and 250 mg/liter free chlorine and with 4 h contact time, MOS exhibits a higher efficacy in oocyst inactivation than hypochlorite. Other RNA-based viability assays, aimed at monitoring the levels of β-tubulin mRNA and 18S rRNA, showed relatively slow decay rates of these molecules following disinfection by chlorine-based oxidants, rendering these molecular diagnostic viability markers inappropriate for disinfection efficacy assessment.
Entry of
Spiroplasma citri
into
Circulifer haematoceps
Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin
Authors: Labroussaa Fabien , Arricau-Bouvery Nathalie , Dubrana Marie-Pierre , Saillard Colette 
Published: January 2010

ABSTRACT

Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between
Spiroplasma citri
and its leafhopper vector,
Circulifer haematoceps
. An
in vitro
protein overlay assay identified five significant binding activities between
S. citri
proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An
S. citri
actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His
6
-tagged PGK from
S. citri
or purified PGK from
Saccharomyces cerevisiae
was added prior to the addition of
S. citri
inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits
S. citri
internalization, demonstrating that the process leading to internalization of
S. citri
in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.
Monitoring the Effects of Chiral Pharmaceuticals on Aquatic Microorganisms by Metabolic Fingerprinting
Authors: Wharfe Emma S. , Winder Catherine L. , Jarvis Roger M. , Goodacre Royston 
Published: January 2010

ABSTRACTThe effects of the chiral pharmaceuticals atenolol and propranolol onPseudomonas putida,Pseudomonas aeruginosa,Micrococcus luteus, andBlastomonas natatoriawere investigated. The growth dynamics of exposed cultures were monitored using a Bioscreen instrument. In addition, Fourier-transform infrared (FT-IR) spectroscopy with appropriate chemometrics and high-performance liquid chromatography (HPLC) were employed in order to investigate the phenotypic changes and possible degradation of the drugs in exposed cultures. For the majority of the bacteria studied there was not a statistically significant difference in the organism's phenotype when it was exposed to the different enantiomers or mixtures of enantiomers. In contrast, the pseudomonads appeared to respond differently to propranolol, and the two enantiomers had different effects on the cellular phenotype. This implies that there were different metabolic responses in the organisms when they were exposed to the different enantiomers. We suggest that our findings may indicate that there are widespread effects on aquatic communities in which active pharmaceutical ingredients are present.
Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD
De Novo
Synthesis Pathway
Authors: Dong Wei-Ren , Xiang Li-Xin , Shao Jian-Zhong 
Published: January 2010

ABSTRACT

The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An
Escherichia coli
strain carrying a defect in NAD
de novo
biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of
E. coli
or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue
E. coli
host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As the QAPRTase gene is ubiquitous in species ranging from bacteria to mammals, the potential environmental biosafety problems caused by horizontal gene transfer can be eliminated.
Insights into the Effect of Soil pH on N
2
O and N
2
Emissions and Denitrifier Community Size and Activity
Authors: Čuhel Jiří , Šimek Miloslav , Laughlin Ronnie J. , Bru David , Chèneby Dominique , Watson Catherine J. , Philippot Laurent 
Published: January 2010

ABSTRACT

The objective of this study was to investigate how changes in soil pH affect the N
2
O and N
2
emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H
2
SO
4
solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N
2
O and N
2
emissions using the
15
N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the
narG
,
napA
,
nirS
,
nirK
, and
nosZ
denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N
2
O/(N
2
O + N
2
) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N
2
O production were observed. Denitrification activity and N
2
O emissions measured under laboratory conditions were correlated with N fluxes
in situ
and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from
in situ
N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between
nirS
,
napA
, and
narG
gene copy numbers and the N
2
O/(N
2
O + N
2
) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.
Prevalence ofBacillus anthracis-Like Organisms and Bacteriophages in the Intestinal Tract of the EarthwormEisenia fetida
Authors: Schuch R. , Pelzek A. J. , Kan S. , Fischetti V. A. 
Published: January 2010

ABSTRACTStable infection ofBacillus anthracislaboratory strains with environmental bacteriophages confers survival phenotypes in soil and earthworm intestinal niches (R. Schuch and V. A. Fischetti, PLoS One 4:e6532, 2009). Here, the natural occurrence of two suchB. anthracis-infective bacteriophages, Wip1 and Wip4, was examined in the intestines ofEisenia fetidaearthworms as part of a 6-year longitudinal study at a Pennsylvania forest site. The Wip1 tectivirus was initially dominant before being supplanted by the Wip4 siphovirus, which was then dominant for the next 3 years. In a host range analysis of a wide-ranging group ofBacillusspecies and related organisms, Wip1 and Wip4 were both infective only towardB. anthracisand certainB. cereusstrains. The natural host of Wip4 remained constant for 3 years and was aB. cereusstrain that expressed aB. anthracis-like surface polysaccharide at septal positions on the cell surface. Next, a novel metagenomic approach was used to determine the extent to which suchB. cereus- andB. anthracis-like strains are found in worms from two geographical locations. Three different enrichment strategies were used for metagenomic DNA isolation, based either on the ability ofB. cereussensu lato to form heat-resistant spores, the sensitivity ofB. anthracisto the PlyG lysin, or the selective amplification of environmental phages cocultured withB. anthracis. Findings from this work indicate thatB. cereussensu lato and its phages are common inhabitants of earthworm intestines.
Metabolic Flux Control at the Pyruvate Node in an AnaerobicEscherichia coliStrain with an Active Pyruvate Dehydrogenase
Authors: Wang Qingzhao , Ou Mark S. , Kim Y. , Ingram L. O. , Shanmugam K. T. 
Published: January 2010

ABSTRACTDuring anaerobic growth ofEscherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant ofE. coliwith a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In apflmutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvatein vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. Apflmutant with LDH and PDH* activities, as well as apfl ldhdouble mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (YATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lowerYATPsuggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering ofE. coliand related bacteria for the production of various biobased products at high rates and yields.