bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of sinusoidal and canalicular elimination of bilirubin‐glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes
Authors: Lengyel György , Veres Zsuzsa , Tugyi Regina , Vereczkey László , Molnár Tünde , Glavinas Hristos , Krajcsi Péter , Jemnitz Katalin
Published: September 2007

Aim: Drug‐induced hyperbilirubinemia has been shown to often be derived from modulation of the expression and activity of hepatobiliary transporters. In this study we examined the interactions of some therapeutic agents, which have been shown to cause cholestasis, with the elimination of bilirubin‐glucuronides, in order to clarify whether these drugs modify the activity of Mrp2 and Mrp3 directly.

Methods: The modulation of bilirubin‐glucuronide elimination with rifampicin, probenecid, indomethacin and benzbromarone was assayed in sandwich cultured rat hepatocytes.

Results: All the drugs studied decreased the canalicular transport, but modified the sinusoidal efflux differently. Rifampicin and probenecid stimulated the sinusoidal efflux, shifting the elimination of bilirubin‐glucuronides to the sinusoidal domain (biliary excretion index: 3.9 ± 1.2; 22.7 ± 7.4 vs. 56.6 ± 1.5 and 56.8 ± 5.5). However, the overall elimination of bilirubin‐glucuronides did not change significantly. In contrast, indomethacin and benzbromarone inhibited bothtransport processes, resulting in the decrease of the overall bilirubin‐glucuronide elimination (61 ± 22; 56 ± 5% of the control). Rifampicin, indomethacin and benzbromarone decreased 5,(6)‐carboxy‐2′,7′‐dichlorofluorescein transport by multidrug resistance‐associated protein (Mrp)2 as visualized by confocal laser microscopy and in vesicular transport experiments. Interestingly, rifampicin decreased the MRP3 activity in vesicular transport experiments using 17‐beta‐estradiol‐17‐beta‐D‐glucuronide as substrate, in contrast to that observed in bilirubin‐glucuronide transport experiments.

Conclusion: Here we show that the interactions of drugs on hepatobiliary transporter proteins may be identifiedin vitroin a sandwich culture of hepatocytes, in which canalicular and sinusoidal transport can be studied separately.
New and Fast Method To Quantify Respiration Rates of Bacterial and Plankton Communities in Freshwater Ecosystems by Using Optical Oxygen Sensor Spots
Authors: Warkentin Mareike , Freese Heike M. , Karsten Ulf , Schumann Rhena
Published: September 2007

A new method of respiration rate measurement based on oxygen luminescence quenching in sensor spots was evaluated for the first time for aquatic bacterial communities. The commonly used Winkler and Clark electrode methods to quantify oxygen concentration both require long incubation times, and the latter additionally causes signal drift due to oxygen consumption at the cathode. The sensor spots proved to be advantageous over those methods in terms of precise and quick oxygen measurements in natural bacterial communities, guaranteeing a respiration rate estimate during a time interval short enough to neglect variations in organism composition, abundance, and activity. Furthermore, no signal drift occurs during measurements, and respiration rate measurements are reliable even at low temperatures and low oxygen consumption rates. Both a natural bacterioplankton sample and a bacterial isolate from a eutrophic river were evaluated in order to optimize the new method for aquatic microorganisms. A minimum abundance of 2.2 × 10
6
respiring cells ml
−1
of a bacterial isolate was sufficient to obtain a distinct oxygen depletion signal within 20 min at 20°C with the new oxygen sensor spot method. Thus, a culture of a bacterial isolate from a eutrophic river (OW 144; 20 × 10
6
respiring bacteria ml
−1
) decreased the oxygen saturation about 8% within 20 min. The natural bacterioplankton sample respired 2.8% from initially 94% oxygen-saturated water in 30 min. During the growth season in 2005, the planktonic community of a eutrophic river consumed between 0.7 and 15.6 μmol O
2
liter
−1
h
−1
. The contribution of bacterial respiration to the total plankton community oxygen consumption varied seasonally between 11 and 100%.
Impact of pH on Lactate Formation and Utilization by Human Fecal Microbial Communities
Authors: Belenguer Alvaro , Duncan Sylvia H. , Holtrop Grietje , Anderson Susan E. , Lobley Gerald E. , Flint Harry J.
Published: September 2007

The human intestine harbors both lactate-producing and lactate-utilizing bacteria. Lactate is normally present at <3 mmol liter−1in stool samples from healthy adults, but concentrations up to 100 mmol liter−1have been reported in gut disorders such as ulcerative colitis. The effect of different initial pH values (5.2, 5.9, and 6.4) upon lactate metabolism was studied with fecal inocula from healthy volunteers, in incubations performed with the addition ofdl-lactate, a mixture of polysaccharides (mainly starch), or both. Propionate and butyrate formation occurred at pH 6.4; both were curtailed at pH 5.2, while propionate but not butyrate formation was inhibited at pH 5.9. With the polysaccharide mix, lactate accumulation occurred only at pH 5.2, but lactate production, estimated usingl-[U-13C]lactate, occurred at all three pH values. Lactate was completely utilized within 24 h at pH 5.9 and 6.4 but not at pH 5.2. At pH 5.9, more butyrate than propionate was formed froml-[U-13C]lactate in the presence of polysaccharides, but propionate, formed mostly by the acrylate pathway, was the predominant product with lactate alone. Fluorescent in situ hybridization demonstrated that populations ofBifidobacteriumspp., major lactate producers, increased approximately 10-fold in incubations with polysaccharides. Populations ofEubacterium hallii, a lactate-utilizing butyrate-producing bacterium, increased 100-fold at pH 5.9 and 6.4. These experiments suggest that lactate is rapidly converted to acetate, butyrate, and propionate by the human intestinal microbiota at pH values as low as 5.9, but at pH 5.2 reduced utilization occurs while production is maintained, resulting in lactate accumulation.
Respiratory Succession and Community Succession of Bacterioplankton in Seasonally Anoxic Estuarine Waters
Authors: Crump Byron C. , Peranteau Cherie , Beckingham Barbara , Cornwell Jeffrey C.
Published: September 2007

Anoxia occurs in bottom waters of stratified estuaries when respiratory consumption of oxygen, primarily by bacteria, outpaces atmospheric and photosynthetic reoxygenation. Once water becomes anoxic, bacterioplankton must change their metabolism to some form of anaerobic respiration. Analysis of redox chemistry in water samples spanning the oxycline of Chesapeake Bay during the summer of 2004 suggested that there was a succession of respiratory metabolism following the loss of oxygen. Bacterial community doubling time, calculated from bacterial abundance (direct counts) and production (anaerobic leucine incorporation), ranged from 0.36 to 0.75 day and was always much shorter than estimates of the time that the bottom water was anoxic (18 to 44 days), indicating that there was adequate time for bacterial community composition to shift in response to changing redox conditions. However, community composition (as determined by PCR-denaturing gradient gel electrophoresis analysis of 16S rRNA genes) in anoxic waters was very similar to that in surface waters in June when nitrate respiration was apparent in the water column and only partially shifted away from the composition of the surface community after nitrate was depleted. Anoxic water communities did not change dramatically until August, when sulfate respiration appeared to dominate. Surface water populations that remained dominant in anoxic waters were
Synechococcus
sp.,
Gammaproteobacteria
in the SAR86 clade, and
Alphaproteobacteria
relatives of
Pelagibacter ubique
, including a putative estuarine-specific
Pelagibacter
cluster. Populations that developed in anoxic water were most similar (<92% similarity) to uncultivated
Firmicutes
, uncultivated
Bacteroidetes, Gammaproteobacteria
in the genus
Thioalcalovibrio
, and the uncultivated SAR406 cluster. These results indicate that typical estuarine bacterioplankton switch to anaerobic metabolism under anoxic conditions but are ultimately replaced by different organisms under sulfidic conditions.
Low-Load Compression Testing: a Novel Way of Measuring Biofilm Thickness
Authors: Paramonova Ekaterina , de Jong Ed D. , Krom Bastiaan P. , van der Mei Henny C. , Busscher Henk J. , Sharma Prashant K.
Published: September 2007

Biofilms are complex and dynamic communities of microorganisms that are studied in many fields due to their abundance and economic impact. Biofilm thickness is an important parameter in biofilm characterization. Current methods of measuring biofilm thicknesses have several limitations, including application, availability, and costs. Here, we present low-load compression testing (LLCT) as a new method for measuring biofilm thickness. With LLCT, biofilm thicknesses are measured during compression by inducing small loads, up to 5 Pa, corresponding to 0.1% deformation, making LLCT essentially a nondestructive technique. Comparison of the thicknesses of various bacterial and yeasts biofilms obtained by LLCT and by using confocal laser scanning microscopy (CLSM) resulted in the conclusion that CLSM underestimates the biofilm thickness due to poor penetration of different fluorescent dyes, especially through the thicker biofilms, whereas LLCT does not suffer from this thickness limitation.
Detection ofSalmonellaspp. in Retail Raw Food Samples from Vietnam and Characterization of Their Antibiotic Resistance
Authors: Van Thi Thu Hao , Moutafis George , Istivan Taghrid , Tran Linh Thuoc , Coloe Peter J.
Published: September 2007

A study was conducted to examine the levels ofSalmonellaspp. contamination in raw food samples, including chicken, beef, pork, and shellfish, from Vietnam and to determine their antibiotic resistance characteristics. A total of 180 samples were collected and examined for the presence ofSalmonellaspp., yielding 91Salmonellaisolates. Sixty-one percent of meat and 18% of shellfish samples were contaminated withSalmonellaspp. Susceptibility of all isolates to a variety of antimicrobial agents was tested, and resistance to tetracycline, ampicillin/amoxicillin, nalidixic acid, sulfafurazole, and streptomycin was found in 40.7%, 22.0%, 18.7%, 16.5%, and 14.3% of the isolates, respectively. Resistance to enrofloxacin, trimethoprim, chloramphenicol, kanamycin, and gentamicin was also detected (8.8 to 2.2%). About half (50.5%) of the isolates were resistant to at least one antibiotic, and multiresistantSalmonellaisolates, resistant to at least three different classes of antibiotics, were isolated from all food types. One isolate from chicken (serovar Albany) contained a variant of theSalmonellagenomic island 1 antibiotic resistance gene cluster. The results show that antibiotic resistance inSalmonellaspp. in raw food samples from Vietnam is significant.
Transcriptomics-Based Identification of Novel Factors Enhancing Heterologous Protein Secretion in Yeasts
Authors: Gasser Brigitte , Sauer Michael , Maurer Michael , Stadlmayr Gerhard , Mattanovich Diethard
Published: September 2007

Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a
Pichia pastoris
strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective
Saccharomyces cerevisiae
homologs of these genes, including the previously characterized secretion helpers
PDI1
,
ERO1
,
SSO2
,
KAR2
/BiP, and
HAC1
as positive controls, were cloned and overexpressed in a
P. pastoris
strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of
P. pastoris
.
Substrate Degradation Kinetics, Microbial Diversity, and Current Efficiency of Microbial Fuel Cells Supplied with Marine Plankton
Authors: Reimers Clare E. , Stecher Hilmar A. , Westall John C. , Alleau Yvan , Howell Kate A. , Soule Leslie , White Helen K. , Girguis Peter R.
Published: September 2007

The decomposition of marine plankton in two-chamber, seawater-filled microbial fuel cells (MFCs) has been investigated and related to resulting chemical changes, electrode potentials, current efficiencies, and microbial diversity. Six experiments were run at various discharge potentials, and a seventh served as an open-circuit control. The plankton consisted of a mixture of freshly captured phytoplankton and zooplankton (0.21 to 1 mm) added at an initial batch concentration of 27.5 mmol liter
−1
particulate organic carbon (OC). After 56.7 days, between 19.6 and 22.2% of the initial OC remained, sulfate reduction coupled to OC oxidation accounted for the majority of the OC that was degraded, and current efficiencies (of the active MFCs) were between 11.3 and 15.5%. In the open-circuit control cell, anaerobic plankton decomposition (as quantified by the decrease in total OC) could be modeled by three terms: two first-order reaction rate expressions (0.79 day
−1
and 0.037 day
−1
, at 15°C) and one constant, no-reaction term (representing 10.6% of the initial OC). However, in each active MFC, decomposition rates increased during the third week, lagging just behind periods of peak electricity generation. We interpret these decomposition rate changes to have been due primarily to the metabolic activity of sulfur-reducing microorganisms at the anode, a finding consistent with the electrochemical oxidization of sulfide to elemental sulfur and the elimination of inhibitory effects of dissolved sulfide. Representative phylotypes, found to be associated with anodes, were allied with
Delta
-,
Epsilon
-, and
Gammaproteobacteria
as well as the
Flavobacterium-Cytophaga-Bacteroides
and
Fusobacteria
. Based upon these results, we posit that higher current efficiencies can be achieved by optimizing plankton-fed MFCs for direct electron transfer from organic matter to electrodes, including microbial precolonization of high-surface-area electrodes and pulsed flowthrough additions of biomass.
Damage in
Escherichia coli
Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature
Authors: Moussa Marwen , Perrier-Cornet Jean-Marie , Gervais Patrick
Published: September 2007

The relationship between membrane permeability, changes in ultrastructure, and inactivation in
Escherichia coli
strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of
E. coli
cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.
Decarboxylation of Sorbic Acid by Spoilage Yeasts Is Associated with the
PAD1
Gene
Authors: Stratford Malcolm , Plumridge Andrew , Archer David B.
Published: September 2007

The spoilage yeast
Saccharomyces cerevisiae
degraded the food preservative sorbic acid (2,4-hexadienoic acid) to a volatile hydrocarbon, identified by gas chromatography mass spectrometry as 1,3-pentadiene. The gene responsible was identified as
PAD1
, previously associated with the decarboxylation of the aromatic carboxylic acids cinnamic acid, ferulic acid, and coumaric acid to styrene, 4-vinylguaiacol, and 4-vinylphenol, respectively. The loss of
PAD1
resulted in the simultaneous loss of decarboxylation activity against both sorbic and cinnamic acids. Pad1p is therefore an unusual decarboxylase capable of accepting both aromatic and aliphatic carboxylic acids as substrates. All members of the
Saccharomyces
genus (sensu stricto) were found to decarboxylate both sorbic and cinnamic acids.
PAD1
homologues and decarboxylation activity were found also in
Candida albicans, Candida dubliniensis, Debaryomyces hansenii
, and
Pichia anomala
. The decarboxylation of sorbic acid was assessed as a possible mechanism of resistance in spoilage yeasts. The decarboxylation of either sorbic or cinnamic acid was not detected for
Zygosaccharomyces, Kazachstania
(
Saccharomyces
sensu lato),
Zygotorulaspora
, or
Torulaspora
, the genera containing the most notorious spoilage yeasts. Scatter plots showed no correlation between the extent of sorbic acid decarboxylation and resistance to sorbic acid in spoilage yeasts. Inhibitory concentrations of sorbic acid were almost identical for
S. cerevisiae
wild-type and Δ
pad1
strains. We concluded that Pad1p-mediated sorbic acid decarboxylation did not constitute a significant mechanism of resistance to weak-acid preservatives by spoilage yeasts, even if the decarboxylation contributed to spoilage through the generation of unpleasant odors.
Yeast Community Structures and Dynamics in Healthy andBotrytis-Affected Grape Must Fermentations
Authors: Nisiotou Aspasia A. , Spiropoulos Apostolos E. , Nychas George-John E.
Published: September 2007

Indigenous yeast population dynamics during the fermentation of healthy andBotrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases,Botrytisinfection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations,Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such asZygosaccharomyces bailiiandIssatchenkiaspp. orKluyveromyces dobzhanskiiandKazachstaniasp. populations that have not been reported before for wine fermentations. Importantly,Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e.,Saccharomyces cerevisiaein samples from Arcadia andZ. bailiiin samples from Attica, could be recovered at the end ofBotrytis-affected fermentations. The governing of wine fermentations byZ. bailiiis reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy andBotrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.
New Insights into the Ligninolytic Capability of a Wood Decay Ascomycete
Authors: Shary Semarjit , Ralph Sally A. , Hammel Kenneth E.
Published: September 2007

Wood-grown cultures of
Daldinia concentrica
oxidized a permethylated β-
14
C-labeled synthetic lignin to
14
CO
2
and also cleaved a permethylated α-
13
C-labeled synthetic lignin to give C
α
-C
β
cleavage products that were detected by
13
C nuclear magnetic resonance spectrometry. Therefore, this ascomycete resembles white-rot basidiomycetes in attacking the recalcitrant nonphenolic structures that predominate in lignin.
Direct Evidence for Production of Microcystins by
Anabaena
Strains from the Baltic Sea
Authors: Halinen Katrianna , Jokela Jouni , Fewer David P. , Wahlsten Matti , Sivonen Kaarina
Published: September 2007

Anabaena
is a filamentous, N
2
-fixing, and morphologically diverse genus of cyanobacteria found in freshwater and brackish water environments worldwide. It contributes to the formation of toxic blooms in freshwater bodies through the production of a range of hepatotoxins or neurotoxins. In the Baltic Sea,
Anabaena
spp. form late summer blooms, together with
Nodularia spumigena
and
Aphanizomenon flos-aquae
. It has been long suspected that Baltic Sea
Anabaena
may produce microcystins. The presence of microcystins has been reported for the coastal regions of the Baltic proper, and a recent report also indicated the presence of the toxin in the open Gulf of Finland. However, at present there is no direct evidence linking Baltic Sea
Anabaena
spp. to microcystin production. Here we report on the isolation of microcystin-producing strains of the genus
Anabaena
in the open Gulf of Finland. The dominant microcystin variants produced by these strains included the highly toxic MCYST-LR as well as [
d
-Asp
3
]MCYST-LR, [
d
-Asp
3
]MCYST-HtyR, MCYST-HtyR, [
d
-Asp
3
,Dha
7
]MCYST-HtyR, and [Dha
7
]MCYST-HtyR variants. Toxic strains were isolated from the coastal Gulf of Finland as well as from the easternmost open-sea sampling station, where there were lower salinities than at other stations. This result suggests that lower salinity may favor microcystin-producing
Anabaena
strains. Furthermore, we sequenced 16S rRNA genes and found evidence for pronounced genetic heterogeneity of the microcystin-producing
Anabaena
strains. Future studies should take into account the potential presence of microcystin-producing
Anabaena
sp. in the Gulf of Finland.
Construction of p16S
lux
, a Novel Vector for Improved Bioluminescent Labeling of Gram-Negative Bacteria
Authors: Riedel Christian U. , Casey Pat G. , Mulcahy Heidi , O'Gara Fergal , Gahan Cormac G. M. , Hill Colin
Published: September 2007

A novel vector has been constructed for the constitutive luminescent tagging of gram-negative bacteria by site-specific integration into the 16S locus of the bacterial chromosome. A number of gram-negative pathogens were successfully tagged using this vector, and the system was validated during murine infections of living animals.
Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their
Flavobacterium
Hosts
Authors: Holmfeldt Karin , Middelboe Mathias , Nybroe Ole , Riemann Lasse
Published: September 2007

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three
Bacteroidetes
strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of
Cellulophaga baltica
(
Flavobacteriaceae
). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.
Influx of Enterococci and Associated Antibiotic Resistance and Virulence Genes from Ready-To-Eat Food to the Human Digestive Tract
Authors: Macovei Lilia , Zurek Ludek
Published: September 2007

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar (∼10
3
CFU/g); the most prevalent were
Enterococcus casseliflavus
(41.5% of isolates) and
Enterococcus hirae
(41.5%) in WI and
Enterococcus faecium
(36.8%),
E. casseliflavus
(27.6%), and
Enterococcus faecalis
(22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common
tet
gene was
tet
(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (
gelE
,
asa1
,
cylA
, and
esp
) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (≤12.3%). Genotyping of
E. faecalis
and
E. faecium
using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of
tet
genes alone to the human digestive tract is 3.8 × 10
5
per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract.
Impact of Plant Functional Group, Plant Species, and Sampling Time on the Composition of
nirK
-Type Denitrifier Communities in Soil
Authors: Bremer Christina , Braker Gesche , Matthies Diethart , Reuter Andreas , Engels Christof , Conrad Ralf
Published: September 2007

We studied the influence of eight nonleguminous grassland plant species belonging to two functional groups (grasses and forbs) on the composition of soil denitrifier communities in experimental microcosms over two consecutive years. Denitrifier community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified
nirK
gene fragments coding for the copper-containing nitrite reductase. The impact of experimental factors (plant functional group, plant species, sampling time, and interactions between them) on the structure of soil denitrifier communities (i.e., T-RFLP patterns) was analyzed by canonical correspondence analysis. While the functional group of a plant did not affect
nirK
-type denitrifier communities, plant species identity did influence their composition. This effect changed with sampling time, indicating community changes due to seasonal conditions and a development of the plants in the microcosms. Differences in total soil nitrogen and carbon, soil pH, and root biomass were observed at the end of the experiment. However, statistical analysis revealed that the plants affected the
nirK
-type denitrifier community composition directly, e.g., through root exudates. Assignment of abundant T-RFs to cloned
nirK
sequences from the soil and subsequent phylogenetic analysis indicated a dominance of yet-unknown
nirK
genotypes and of genes related to
nirK
from denitrifiers of the order
Rhizobiales
. In conclusion, individual species of nonleguminous plants directly influenced the composition of denitrifier communities in soil, but environmental conditions had additional significant effects.
Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in
Giardia intestinalis
Authors: Dawson Scott C. , Sagolla Meredith S. , Mancuso Joel J. , Woessner David J. , House Susan A. , Fritz-Laylin Lillian , Cande W. Zacheus
Published: September 2007

Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in
Giardia intestinalis
, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in
Giardia
. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in
Giardia
and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.
Candida albicans
Sfl1 Suppresses Flocculation and Filamentation
Authors: Bauer Janine , Wendland Jürgen
Published: September 2007

Hyphal morphogenesis in
Candida albicans
is regulated by multiple pathways which act by either inducing or repressing filamentation. Most notably, Tup1, Nrg1, and Rfg1 are transcriptional repressors, while Efg1, Flo8, Cph1, and Czf1 can induce filamentation. Here, we present the functional analysis of Ca
SFL1
, which encodes the
C. albicans
homolog of the
Saccharomyces cerevisiae SFL1
(suppressor of flocculation) gene. Deletion of Ca
SFL1
results in flocculation (i.e., the formation of clumps) of yeast cells, which is most pronounced in minimal medium. The flocs contained hyphae already under noninducing conditions, and filamentation could be enhanced with hypha-inducing cues at 37°C. Expression of
SFL1
in a heterozygous mutant under the control of the Ca
MET3
promoter was shown to complement these defects and allowed switching between wild-type and mutant phenotypes. Interestingly, increased expression of
SFL1
using a
MET3prom-SFL1
construct prior to the induction of filamentation completely blocked germ tube formation. To localize Sfl1 in vivo, we generated a
SFL1-GFP
fusion. Sfl1-green fluorescent protein was found in the nucleus in both yeast cells and, to a lesser extent, hyphal cells. Using reverse transcription-PCR, we find an increased expression of
ALS1, ALS3, HWP1, ECE1
, and also
FLO8
. Our results suggest that Sfl1 functions in the repression of flocculation and filamentation and thus represents a novel negative regulator of
C. albicans
morphogenesis.
Transcriptional Gene Silencing Reveals Two Distinct Groups of
Entamoeba histolytica
Gal/GalNAc-Lectin Light Subunits
Authors: Bracha Rivka , Nuchamowitz Yael , Wender Nomy , Mirelman David
Published: September 2007

The
Entamoeba histolytica
cell surface Gal/GalNAc-inhibitable lectin is a heterodimer between a heavy (170 kDa) subunit linked via a disulfide bond to a light (31 to 35 kDa) subunit. Five light subunit genes with high homology have been identified (Eh
lgl1
to -
5
). We have previously shown that silencing of the expression of Eh
lgl1
, in the G3 trophozoites which had already been silenced in the amoebapore gene (Eh
ap-a
), also suppressed the transcription of Eh
lgl2
and -
3
(strain RBV). The total absence of the lgl1 to -3 subunits in the RBV trophozoites affected their ability to cap the surface Gal-lectin molecules to the uroid region. We have now found that in the RBV trophozoites, the lgl4 and -5 subunits (31 kDa) are overexpressed and appear to compensate for the missing lgl1 to -3 in the heterodimer complex. Transcriptional silencing of Eh
lgl5
was achieved by transfection of G3 trophozoites with a plasmid containing the open reading frame of Eh
lgl5
ligated to the 5′ promoter region of the Eh
ap-a
gene. The transfected trophozoites (strain L5) were silenced in Eh
lgl5
and the closely related Eh
lgl4
, while the expression of the larger lgl1 to -3 subunits was upregulated. L5 trophozoites retained their ability to cap the Gal-lectin molecules. Attempts to simultaneously silence all of the Eh
lgl
genes have failed so far, possibly due to their crucial importance to the Gal-lectin functions. Our ability to silence part of the genes belonging to the same family can serve as a tool to study the relationships and functions of the members of other gene families.
Hydrogen Production by Termite Gut Protists: Characterization of Iron Hydrogenases of Parabasalian Symbionts of the Termite
Coptotermes formosanus
Authors: Inoue Jun-Ichi , Saita Kanako , Kudo Toshiaki , Ui Sadaharu , Ohkuma Moriya
Published: September 2007

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H
2
) that is emitted by the termites; however, little is known about the physiology and biochemistry of H
2
production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H
2
production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in
Pseudotrichonympha grassii
, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite
Coptotermes formosanus
. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H
2
evolution rather than H
2
uptake. H
2
evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H
2
evolution activity was detected in the hydrogenosomal fraction of
P. grassii
cells; however, the vigorous H
2
uptake activity of the endosymbiotic bacteria compensated for the strong H
2
evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H
2
production in termite guts has been largely underestimated.
Fully Codon-Optimized
luciferase
Uncovers Novel Temperature Characteristics of the
Neurospora
Clock
Authors: Gooch Van D. , Mehra Arun , Larrondo Luis F. , Fox Julie , Touroutoutoudis Melissa , Loros Jennifer J. , Dunlap Jay C.
Published: September 2007

We report the complete reconstruction of the firefly
luciferase
gene, fully codon optimized for expression in
Neurospora crassa
. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the
frq
or
eas/ccg-2
promoter is used to drive
luciferase
, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the
frq
promoter drives
luciferase
exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.
Motility and Chemotaxis in
Agrobacterium tumefaciens
Surface Attachment and Biofilm Formation
Authors: Merritt Peter M. , Danhorn Thomas , Fuqua Clay
Published: September 2007

Bacterial motility mechanisms, including swimming, swarming, and twitching, are known to have important roles in biofilm formation, including colonization and the subsequent expansion into mature structured surface communities. Directed motility requires chemotaxis functions that are conserved among many bacterial species. The biofilm-forming plant pathogen
Agrobacterium tumefaciens
drives swimming motility by utilizing a small group of flagella localized to a single pole or the subpolar region of the cell. There is no evidence for twitching or swarming motility in
A. tumefaciens.
Site-specific deletion mutations that resulted in either aflagellate, flagellated but nonmotile, or flagellated but nonchemotactic
A. tumefaciens
derivatives were examined for biofilm formation under static and flowing conditions. Nonmotile mutants were significantly deficient in biofilm formation under static conditions. Under flowing conditions, however, the aflagellate mutant rapidly formed aberrantly dense, tall biofilms. In contrast, a nonmotile mutant with unpowered flagella was clearly debilitated for biofilm formation relative to the wild type. A nontumbling chemotaxis mutant was only weakly affected with regard to biofilm formation under nonflowing conditions but was notably compromised in flow, generating less adherent biomass than the wild type, with a more dispersed cellular arrangement. Extragenic suppressor mutants of the chemotaxis-impaired, straight-swimming phenotype were readily isolated from motility agar plates. These mutants regained tumbling at a frequency similar to that of the wild type. Despite this phenotype, biofilm formation by the suppressor mutants in static cultures was significantly deficient. Under flowing conditions, a representative suppressor mutant manifested a phenotype similar to yet distinct from that of its nonchemotactic parent.
Virulence and Prodigiosin Antibiotic Biosynthesis inSerratiaAre Regulated Pleiotropically by the GGDEF/EAL Domain Protein, PigX
Authors: Fineran Peter C. , Williamson Neil R. , Lilley Kathryn S. , Salmond George P. C.
Published: September 2007

Gram-negative bacteria of the genusSerratiaare opportunistic human, plant, and insect pathogens.Serratiasp. strain ATCC 39006 secretes pectinases and cellulases and produces the secondary metabolites carbapenem and prodigiosin. Mutation of a gene (pigX) resulted in an extremely pleiotropic phenotype: prodigiosin antibiotic biosynthesis, plant virulence, and pectinase production were all elevated. PigX controlled secondary metabolism by repressing the transcription of the target prodigiosin biosynthetic operon (pigA-pigO). The transcriptional start site ofpigXwas determined, andpigXexpression occurred in parallel with Pig production. Detailed quantitative intracellular proteome analyses enabled the identification of numerous downstream targets of PigX, including OpgG, mutation of which reduced the production of the plant cell wall-degrading enzymes and virulence. The highly pleiotropic PigX regulator contains GGDEF and EAL domains with noncanonical motifs and is predicted to be membrane associated. Genetic evidence suggests that PigX might function as a cyclic dimeric GMP phosphodiesterase. This is the first characterization of a GGDEF and EAL domain protein inSerratiaand the first example of the regulation of antibiotic production by a GGDEF/EAL domain protein.
Antigen 84, an Effector of Pleiomorphism inMycobacterium smegmatis
Authors: Nguyen Liem , Scherr Nicole , Gatfield John , Walburger Anne , Pieters Jean , Thompson Charles J.
Published: September 2007

While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such asMycobacteriumspecies are unknown. Here we show that aMycobacterium smegmatisprotein homologous to eubacterial DivIVA-like proteins, includingM. tuberculosisantigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84,wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches.M. bovisBCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.
Strain-Ecotype Specificity in
Sinorhizobium meliloti
-
Medicago truncatula
Symbiosis Is Correlated to Succinoglycan Oligosaccharide Structure
Authors: Simsek Senay , Ojanen-Reuhs Tuula , Stephens Samuel B. , Reuhs Bradley L.
Published: September 2007

Molecular signals, including Nod factors and succinoglycan, are necessary for the establishment of nitrogen-fixing nodules (Fix
+
) in
Medicago truncatula
-
Sinorhizobium meliloti
symbiosis. This report shows that
M. truncatula
-
S. meliloti
interactions involve ecotype-strain specificity, as
S. meliloti
Rm41 and NRG247 are Fix
+
(compatible) on
M. truncatula
A20 and Fix
−
(incompatible) on
M. truncatula
A17, the Fix phenotypes are reversed with
S. meliloti
NRG185 and NRG34, and there is a correlation between the host specificity and succinoglycan oligosaccharide structure.
S. meliloti
NRG185 produces oligosaccharides that are almost fully succinylated, with two succinate groups per subunit, whereas the oligosaccharides produced by
S. meliloti
Rm41 include many nonsuccinylated subunits, as well as subunits with a single succinate group and others with malate. The results of this study demonstrated the following: (i) incompatibility is not a consequence of an avirulence factor or lack of Nod factor activity; (ii) the Fix
+
phenotypes are succinoglycan dependent; (iii) there is structural variability in the succinoglycan oligosaccharide populations between
S. meliloti
strains; (iv) the structural nature of the succinoglycan oligosaccharides is correlated to compatibility; most importantly, (v) an
S. meliloti
Rm41 derivative, carrying
exo
genes from an
M. truncatula
A17-compatible strain, produced a modified population of succinoglycan oligosaccharides (similar to the donor strain) and was Fix
+
on A17.
Differential Roles of Individual Domains in Selection of Secretion Route of a
Streptococcus parasanguinis
Serine-Rich Adhesin, Fap1
Authors: Chen Qiang , Sun Baiming , Wu Hui , Peng Zhixiang , Fives-Taylor Paula M.
Published: September 2007

Fimbria-associated protein 1 (Fap1) is a high-molecular-mass glycosylated surface adhesin required for fimbria biogenesis and biofilm formation in
Streptococcus parasanguinis
. The secretion of mature Fap1 is dependent on the presence of SecA2, a protein with some homology to, but with a different role from, SecA. The signals that direct the secretion of Fap1 to the SecA2-dependent secretion pathway rather than the SecA-dependent secretion pathway have not yet been identified. In this study, Fap1 variants containing different domains were expressed in both
secA2
wild-type and mutant backgrounds and were tested for their ability to be secreted by the SecA- or SecA2-dependent pathway. The presence or absence of the cell wall anchor domain (residues 2531 to 2570) at the C terminus did not alter the selection of the Fap1 secretion route. The Fap1 signal peptide (residues 1 to 68) was sufficient to support the secretion of a heterologous protein via the SecA-dependent pathway, suggesting that the signal peptide was sufficient for recognition by the SecA-dependent pathway. The minimal sequences of Fap1 required for the SecA2-dependent pathway included the N-terminal signal peptide, nonrepetitive region I (residues 69 to 102), and part of nonrepetitive region II (residues 169 to 342). The two serine-rich repeat regions (residues 103 to 168 and 505 to 2530) were not required for Fap1 secretion. However, they were both involved in the specific inhibition of Fap1 secretion via the SecA-dependent pathway.