bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation ofLactobacillus caseiSorbitol Utilization Genes Requires DNA-Binding Transcriptional Activator GutR and the Conserved Protein GutM
Authors: Alcántara Cristina , Sarmiento-Rubiano Luz Adriana , Monedero Vicente , Deutscher Josef , Pérez-Martínez Gaspar , Yebra María J.
Published: August 2008

Sequence analysis of the five genes (gutRMCBA) downstream from the previously described sorbitol-6-phosphate dehydrogenase-encodingLactobacillus casei gutFgene revealed that they constitute a sorbitol (glucitol) utilization operon. ThegutRMgenes encode putative regulators, while thegutCBAgenes encode the EIIC, EIIBC, and EIIA proteins of a phosphoenolpyruvate-dependent sorbitol phosphotransferase system (PTSGut). Thegutoperon is transcribed as a polycistronicgutFRMCBAmessenger, the expression of which is induced by sorbitol and repressed by glucose.gutRencodes a transcriptional regulator with two PTS-regulated domains, a galactitol-specific EIIB-like domain (EIIBGatdomain) and a mannitol/fructose-specific EIIA-like domain (EIIAMtldomain). Its inactivation abolishedgutoperon transcription and sorbitol uptake, indicating that it acts as a transcriptional activator. In contrast, cells carrying agutBmutation expressed thegutoperon constitutively, but they failed to transport sorbitol, indicating that EIIBCGutnegatively regulates GutR. A footprint analysis showed that GutR binds to a 35-bp sequence upstream from thegutpromoter. A sequence comparison with the presumed promoter region ofgutoperons from various firmicutes revealed a GutR consensus motif that includes an inverted repeat. The regulation mechanism of theL. casei gutoperon is therefore likely to be operative in other firmicutes. Finally,gutMcodes for a conserved protein of unknown function present in all sequencedgutoperons. AgutMmutant, the first constructed in a firmicute, showed drastically reducedgutoperon expression and sorbitol uptake, indicating a regulatory role also for GutM.
Effects of Hypercapnic Hypoxia on Inactivation and Elimination of
Vibrio campbellii
in the Eastern Oyster,
Crassostrea virginica
Authors: Macey Brett M. , Achilihu Ikenna O. , Burnett Karen G. , Burnett Louis E.
Published: August 2008

The Eastern oyster,
Crassostrea virginica
, inhabits shallow coastal waters that frequently experience periods of low dissolved oxygen (hypoxia) and elevated CO
2
(hypercapnia) levels. Bacteria are extremely abundant in these environments and accumulate in large numbers in filter-feeding oysters, which can act as passive carriers of human pathogens. Although hypercapnic hypoxia (HH) can affect certain specific immune mechanisms, its direct effect on the inactivation, degradation and elimination of bacteria in oysters is unknown. This research was conducted to determine whether exposure to HH reduces the ability of
C. virginica
to inactivate and eliminate
Vibrio campbellii
following its injection into the adductor muscle. Oysters were held in fully air-saturated (normoxic; partial O
2
pressure [P
O2
] = 20.7 kPa, CO
2
< 0.06 kPa, pH 7.8 to 8.0) or HH (P
O2
= 4 kPa, CO
2
= 1.8 kPa, pH 6.5 to 6.8) seawater at 25°C for 4 h before being injected in the adductor muscle with 10
5
live
Vibrio campbellii
bacteria and remained under these conditions for the remainder of the experiment (up to 24 h postinjection). Real-time PCR was used to quantify the number of intact
V. campbellii
bacteria, while selective plating was used to quantify the number of injected bacteria remaining culturable in whole-oyster tissues, seawater, and feces/pseudofeces at 0, 1, 4, and 24 h postinjection. We found that oysters maintained under normoxic conditions were very efficient at inactivating and degrading large numbers of injected bacteria within their tissues. Moreover, a small percentage (∼10%) of injected bacteria were passed into the surrounding seawater, while less than 1% were recovered in the feces/pseudofeces. In contrast, HH increased the percentage of culturable bacteria recovered from the tissues of oysters, suggesting an overall decrease in bacteriostasis. We suggest that poor water quality may increase the risk that oysters will harbor and transmit bacterial pathogens hazardous to human and ecosystem health.
Engineering of an
Escherichia coli
Strain for the Production of 3-Methyl-1-Butanol
Authors: Connor Michael R. , Liao James C.
Published: August 2008

3-Methyl-1-butanol is a potential fuel additive or substitute. Previously this compound was identified in small quantities in yeast fermentation as one of the fusel alcohols. In this work, we engineered an
Escherichia coli
strain to produce 3-methyl-1-butanol from glucose via the host's amino acid biosynthetic pathways. Strain improvement with the removal of feedback inhibition and competing pathways increased the selectivity and productivity of 3-methyl-1-butanol. This work demonstrates the feasibility of production of 3-methyl-1-butanol as a biofuel and shows promise in using
E. coli
as a host for production.
Transformation of
N
-Phenylpiperazine by Mixed Cultures from a Municipal Wastewater Treatment Plant
Authors: Jung Carina M. , Heinze Thomas M. , Deck Joanna , Strakosha Ruth , Sutherland John B.
Published: August 2008

Samples from a wastewater treatment plant were used as inocula for mixed cultures dosed with
N
-phenylpiperazine (NPP), a model compound containing the piperazine ring found in many fluoroquinolones. Chemical analyses showed that NPP (50 mg liter
−1
) disappeared in 12 days, with the appearance of a transient metabolite and two nitrosated compounds.
Assessment of Inhibitory Effects of Fluoride-Coated Tubes on Biofilm Formation by Using the In Vitro Dental Unit Waterline Biofilm Model
Authors: Yabune Toshiaki , Imazato Satoshi , Ebisu Shigeyuki
Published: August 2008

This study aimed to establish an in vitro model to simulate biofilms formed in dental unit waterlines (DUWLs) and to investigate the ability of polyvinylidene fluoride (PVDF)-coated tubes to inhibit biofilm formation using this model. The water and biofilm samples were obtained from DUWLs which had been clinically used for 2.5 years, and the predominant bacteria were identified. A conventional polyurethane tube was incubated for 24 to 96 h in the mixed flora of isolated bacteria, and the optimal incubation conditions to simulate a clinically formed biofilm were determined by observation with a scanning electron microscope. Biofilm formation on a PVDF-coated tube was observed using this in vitro model, and the adherence of different bacterial species to conventional and PVDF-coated tubes was assessed.
Sphingomonas paucimobilis
,
Acinetobacter haemolytics
, and
Methylobacterium mesophilicum
were predominantly isolated from contaminated DUWLs. Incubation of the polyurethane tube with the mixed flora containing these three species for 96 h resulted in the formation of a mature biofilm similar to the one clinically observed. The PVDF-coated tube was significantly less adhesive to all three bacterial species than the polyurethane tube (
P
< 0.05 by the Mann-Whitney U test), and the attachment of small amounts of rods was observed even after incubation with the mixed flora for 96 h. In conclusion, an in vitro biofilm model was obtained by using a mixed flora of bacteria isolated from DUWLs, and the PVDF-coated tube was found to be effective in preventing biofilm formation using this model.
Evaluation of a Low-Cost Electrostatic Dust Fall Collector for Indoor Air Endotoxin Exposure Assessment
Authors: Noss Ilka , Wouters Inge M. , Visser Maaike , Heederik Dick J. J. , Thorne Peter S. , Brunekreef Bert , Doekes Gert
Published: August 2008

Exposure to endotoxin in home environments has become a key issue in asthma and allergy research. Most studies have analyzed floor or mattress dust endotoxin, but its validity as a proxy for airborne exposure is unknown, while active airborne dust sampling is not feasible in large-scale population studies because of logistic and financial limitations. We therefore developed and evaluated a simple passive airborne dust collection method for airborne endotoxin exposure assessment. We explored an electrostatic dust fall collector (EDC), consisting of a 42- by 29.6-cm-sized folder with four electrostatic cloths exposed to the air. The EDC was tested during two 14-day periods in seven nonfarm and nine farm homes and in farm stables. In parallel, active airborne dust sampling was performed with Harvard impactors and floor dust collected by vacuuming, using nylon sampling socks. The endotoxin levels could be measured in all EDC cloth extracts. The levels (in EU/m2) between EDCs used simultaneously or in different sampling periods in the same home correlated strongly (r> 0.8). EDC endotoxin also correlated moderately to strongly (r= 0.6 to 0.8) with the endotoxin measured by active airborne dust sampling and living room floor dust sampling and—in farm homes—with the endotoxin captured by the EDC in stables. In contrast, endotoxin levels measured by floor dust sampling showed only a poor correlation with the levels measured by active airborne dust sampling. We therefore conclude that measuring endotoxin levels with the EDC is a valid measure of average airborne endotoxin exposure, while reproducibility over time is at least equivalent to that of reservoir dust analyses.
Preferential Colonization ofSolanum tuberosumL. Roots by the FungusGlomus intraradicesin Arable Soil of a Potato Farming Area
Authors: Cesaro Patrizia , van Tuinen Diederik , Copetta Andrea , Chatagnier Odile , Berta Graziella , Gianinazzi Silvio , Lingua Guido
Published: August 2008

The symbiosis between plant roots and arbuscular mycorrhizal (AM) fungi has been shown to affect both the diversity and productivity of agricultural communities. In this study, we characterized the AM fungal communities ofSolanum tuberosumL. (potato) roots and of the bulk soil in two nearby areas of northern Italy, in order to verify if land use practices had selected any particular AM fungus with specificity to potato plants. The AM fungal large-subunit (LSU) rRNA genes were subjected to nested PCR, cloning, sequencing, and phylogenetic analyses. One hundred eighty-three LSU rRNA sequences were analyzed, and eight monophyletic ribotypes, belonging toGlomusgroups A and B, were identified. AM fungal communities differed between bulk soil and potato roots, as one AM fungal ribotype, corresponding toGlomus intraradices, was much more frequent in potato roots than in soils (accounting for more than 90% of sequences from potato samples and less than 10% of sequences from soil samples). A semiquantitative heminested PCR with specific primers was used to confirm and quantify the AM fungal abundance observed by cloning. Overall results concerning the biodiversity of AM fungal communities in roots and in bulk soils from the two studied areas suggested that potato roots were preferentially colonized by one AM fungal species,G. intraradices.
Characterization of an Endoglucanase Belonging to a New Subfamily of Glycoside Hydrolase Family 45 of the BasidiomycetePhanerochaete chrysosporium
Authors: Igarashi Kiyohiko , Ishida Takuya , Hori Chiaki , Samejima Masahiro
Published: August 2008

The wood decay fungusPhanerochaete chrysosporiumhas served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeastPichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (<22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley β-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.
Persistence of
Helicobacter pylori
in Heterotrophic Drinking-Water Biofilms
Authors: Gião M. S. , Azevedo N. F. , Wilks S. A. , Vieira M. J. , Keevil C. W.
Published: August 2008

Although the route of transmission of
Helicobacter pylori
remains unknown, drinking water has been considered a possible transmission vector. It has been shown previously that, in water, biofilms are a protective niche for several pathogens, protecting them from stressful conditions, such as low carbon concentration, shear stress, and less-than-optimal temperatures. In this work, the influence of these three parameters on the persistence and cultivability of
H. pylori
in drinking-water biofilms was studied. Autochthonous biofilm consortia were formed in a two-stage chemostat system and then inoculated with the pathogen. Total numbers of
H. pylori
cells were determined by microscopy using a specific
H. pylori
16S rRNA peptide nucleic acid probe, whereas cultivable cells were assessed by standard plating onto selective
H. pylori
medium. Cultivable
H. pylori
could not be detected at any time point, but the ability of
H. pylori
cells to incorporate, undergo morphological transformations, persist, and even agglomerate in biofilms for at least 31 days without a noticeable decrease in the total cell number (on average, the concentration was between 1.54 × 10
6
and 2.25 × 10
6
cells cm
−2
) or in the intracellular rRNA content may indicate that the loss of cultivability was due to entry into a viable but noncultivable state. Unlike previous results obtained for pure-culture
H. pylori
biofilms, shear stress did not negatively influence the numbers of
H. pylori
cells attached, suggesting that the autochthonous aquatic bacteria have an important role in retaining this pathogen in the sessile state, possibly by providing suitable microaerophilic environments or linking biomolecules to which the pathogen adheres. Therefore, biofilms appear to provide not only a safe haven for
H. pylori
but also a concentration mechanism so that subsequent sloughing releases a concentrated bolus of cells that might be infectious and that could escape routine grab sample microbiological analyses and be a cause of concern for public health.
Global Occurrence of Archaeal
amoA
Genes in Terrestrial Hot Springs
Authors: Zhang Chuanlun L. , Ye Qi , Huang Zhiyong , Li WenJun , Chen Jinquan , Song Zhaoqi , Zhao Weidong , Bagwell Christopher , Inskeep William P. , Ross Christian , Gao Lei , Wiegel Juergen , Romanek Christopher S. , Shock Everett L. , Hedlund Brian P.
Published: August 2008

Despite the ubiquity of ammonium in geothermal environments and the thermodynamic favorability of aerobic ammonia oxidation, thermophilic ammonia-oxidizing microorganisms belonging to the crenarchaeota kingdom have only recently been described. In this study, we analyzed microbial mats and surface sediments from 21 hot spring samples (pH 3.4 to 9.0; temperature, 41 to 86°C) from the United States, China, and Russia and obtained 846 putative archaeal ammonia monooxygenase large-subunit (
amoA
) gene and transcript sequences, representing a total of 41
amoA
operational taxonomic units (OTUs) at 2% identity. The
amoA
gene sequences were highly diverse, yet they clustered within two major clades of archaeal
amoA
sequences known from water columns, sediments, and soils: clusters A and B. Eighty-four percent (711/846) of the sequences belonged to cluster A, which is typically found in water columns and sediments, whereas 16% (135/846) belonged to cluster B, which is typically found in soils and sediments. Although a few
amoA
OTUs were present in several geothermal regions, most were specific to a single region. In addition, cluster A
amoA
genes formed geographic groups, while cluster B sequences did not group geographically. With the exception of only one hot spring, principal-component analysis and UPGMA (unweighted-pair group method using average linkages) based on the UniFrac metric derived from cluster A grouped the springs by location, regardless of temperature or bulk water pH, suggesting that geography may play a role in structuring communities of putative ammonia-oxidizing archaea (AOA). The
amoA
genes were distinct from those of low-temperature environments; in particular, pair-wise comparisons between hot spring
amoA
genes and those from sympatric soils showed less than 85% sequence identity, underscoring the distinctness of hot spring archaeal communities from those of the surrounding soil system. Reverse transcription-PCR showed that
amoA
genes were transcribed in situ in one spring and the transcripts were closely related to the
amoA
genes amplified from the same spring. Our study demonstrates the global occurrence of putative archaeal
amoA
genes in a wide variety of terrestrial hot springs and suggests that geography may play an important role in selecting different assemblages of AOA.
Monitoring Abundance and Expression of “
Dehalococcoides
” Species Chloroethene-Reductive Dehalogenases in a Tetrachloroethene-Dechlorinating Flow Column
Authors: Behrens Sebastian , Azizian Mohammad F. , McMurdie Paul J. , Sabalowsky Andrew , Dolan Mark E. , Semprini Lew , Spormann Alfred M.
Published: August 2008

We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified “
Dehalococcoides
” species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (
pceA, tceA, vcrA
, and
bvcA
) in nucleic acid extracts from different sections of the column.
Dehalococcoides
16S rRNA gene copies were highest at the inflow port [(3.6 ± 0.6) × 10
6
(mean ± standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for
tceA, vcrA
, and
bvcA
were detected 5 to 10 cm from the column inflow.
bvcA
was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of
pceA
was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the
Dehalococcoides
population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of
Firmicutes
and
Actinobacteria
. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating
Dehalococcoides
spp. and their role in bioremediation of chloroethenes.
Attached Bacterial Populations Shared by Four Species of Aquatic Angiosperms
Authors: Crump Byron C. , Koch Evamaria W.
Published: August 2008

Symbiotic relationships between microbes and plants are common and well studied in terrestrial ecosystems, but little is known about such relationships in aquatic environments. We compared the phylogenetic diversities of leaf- and root-attached bacteria from four species of aquatic angiosperms using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of PCR-amplified 16S rRNA genes. Plants were collected from three beds in Chesapeake Bay at sites characterized as freshwater (Vallisneria americana), brackish (Potomogeton perfoliatusandStuckenia pectinata), and marine (Zostera marina). DGGE analyses showed that bacterial communities were very similar for replicate samples of leaves from canopy-forming plantsS. pectinataandP. perfoliatusand less similar for replicate samples of leaves from meadow-forming plantsZ. marinaandV. americanaand of roots of all species. In contrast, bacterial communities differed greatly among plant species and between leaves and roots. DNA sequencing identified 154 bacterial phylotypes, most of which were restricted to single plant species. However, 12 phylotypes were found on more than one plant species, and several of these phylotypes were abundant in clone libraries and represented the darkest bands in DGGE banding patterns. Root-attached phylotypes included relatives of sulfur-oxidizingGammaproteobacteriaand sulfate-reducingDeltaproteobacteria. Leaf-attached phylotypes included relatives of polymer-degradingBacteroidetesand phototrophicAlphaproteobacteria. Also, leaves and roots of three plant species hosted relatives of methylotrophicBetaproteobacteriabelonging to the familyMethylophilaceae. These results suggest that aquatic angiosperms host specialized communities of bacteria on their surfaces, including several broadly distributed and potentially mutualistic bacterial populations.
Antibacterial Activities of Naturally Occurring Compounds against
Mycobacterium avium
subsp.
paratuberculosis
Authors: Wong Stella Y. Y. , Grant Irene R. , Friedman Mendel , Elliott Christopher T. , Situ Chen
Published: August 2008

The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of
Mycobacterium avium
subsp.
paratuberculosis
(a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method.
M. avium
subsp.
paratuberculosis
was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 μg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37°C. Six of the compounds were found to inhibit the growth of
M. avium
subsp.
paratuberculosis
. The most effective compound was
trans
-cinnamaldehyde, with a MIC of 25.9 μg/ml, followed by cinnamon oil (26.2 μg/ml), oregano oil (68.2 μg/ml), carvacrol (72.2 μg/ml), 2,5-dihydroxybenzaldehyde (74 μg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 μg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or the aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three
M. avium
subsp.
paratuberculosis
strains studied. Possible mechanisms of the antimicrobial effects are discussed.
Physiological and Transcriptional Responses to High Concentrations of Lactic Acid in Anaerobic Chemostat Cultures of
Saccharomyces cerevisiae
Authors: Abbott Derek A. , Suir Erwin , van Maris Antonius J. A. , Pronk Jack T.
Published: August 2008

Based on the high acid tolerance and the simple nutritional requirements of
Saccharomyces cerevisiae
, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to
S. cerevisiae
, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the
haa1
Δ mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of
S. cerevisiae
to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.
Optimization of Metabolic Capacity and Flux through Environmental Cues To Maximize Hydrogen Production by the Cyanobacterium “
Arthrospira
(
Spirulina
)
maxima
”
Authors: Ananyev Gennady , Carrieri Damian , Dismukes G. Charles
Published: August 2008

Environmental and nutritional conditions that optimize the yield of hydrogen (H
2
) from water using a two-step photosynthesis/fermentation (P/F) process are reported for the hypercarbonate-requiring cyanobacterium “
Arthrospira maxima
.” Our observations lead to four main conclusions broadly applicable to fermentative H
2
production by bacteria: (i) anaerobic H
2
production in the dark from whole cells catalyzed by a bidirectional [NiFe] hydrogenase is demonstrated to occur in two temporal phases involving two distinct metabolic processes that are linked to prior light-dependent production of NADPH (photosynthetic) and dark/anaerobic production of NADH (fermentative), respectively; (ii) H
2
evolution from these reductants represents a major pathway for energy production (ATP) during fermentation by regenerating NAD
+
essential for glycolysis of glycogen and catabolism of other substrates; (iii) nitrate removal during fermentative H
2
evolution is shown to produce an immediate and large stimulation of H
2
, as nitrate is a competing substrate for consumption of NAD(P)H, which is distinct from its slower effect of stimulating glycogen accumulation; (iv) environmental and nutritional conditions that increase anaerobic ATP production, prior glycogen accumulation (in the light), and the intracellular reduction potential (NADH/NAD
+
ratio) are shown to be the key variables for elevating H
2
evolution. Optimization of these conditions and culture age increases the H
2
yield from a single P/F cycle using concentrated cells to 36 ml of H
2
/g (dry weight) and a maximum 18% H
2
in the headspace. H
2
yield was found to be limited by the hydrogenase-mediated H
2
uptake reaction.
Methanogenesis from Methanol at Low Temperatures by a Novel Psychrophilic Methanogen, “
Methanolobus psychrophilus
” sp. nov., Prevalent in Zoige Wetland of the Tibetan Plateau
Authors: Zhang Guishan , Jiang Na , Liu Xiaoli , Dong Xiuzhu
Published: August 2008

The Zoige wetland of the Tibetan plateau is at permanent low temperatures and is a methane emission heartland of the plateau; however, cold-adaptive methanogens in the soil are poorly understood. In this study, a variety of methanogenic enrichments at 15°C and 30°C were obtained from the wetland soil. It was demonstrated that hydrogenotrophic methanogenesis was the most efficient type at 30°C, while methanol supported the highest methanogenesis rate at 15°C. Moreover, methanol was the only substrate to produce methane more efficiently at 15°C than at 30°C. A novel psychrophilic methanogen, strain R15, was isolated from the methanol enrichment at 15°C. Phylogenetic analysis placed strain R15 within the genus
Methanolobus
, loosely clustered with
Methanolobus taylorii
(96.7% 16S rRNA similarity). R15 produced methane from methanol, trimethylamine, and methyl sulfide and differed from other
Methanolobus
species by growing and producing methane optimally at 18°C (specific growth rate of 0.063 ± 0.001 h
−1
) and even at 0°C. Based on these characteristics, R15 was proposed to be a new species and named “
Methanolobus psychrophilus
” sp. nov. The

K
m

and
V
max
of R15 for methanol conversion were determined to be 87.5 ± 0.4 μM and 0.39 ± 0.04 mM h
−1
at 18°C, respectively, indicating a high affinity and conversion efficiency for methanol. The proportion of R15 in the soil was determined by quantitative PCR, and it accounted for 17.2% ± 2.1% of the total archaea, enumerated as 10
7
per gram of soil; the proportion was increased to 42.4% ± 2.3% in the methanol enrichment at 15°C. This study suggests that the psychrophilic methanogens in the Zoige wetland are likely to be methylotrophic and to play a role in methane emission of the wetland.
A New Huanglongbing Species, “
Candidatus
Liberibacter psyllaurous,” Found To Infect Tomato and Potato, Is Vectored by the Psyllid
Bactericera cockerelli
(Sulc)
Authors: Hansen A. K. , Trumble J. T. , Stouthamer R. , Paine T. D.
Published: August 2008

A new huanglongbing (HLB) “
Candidatus
Liberibacter” species is genetically characterized, and the bacterium is designated “
Candidatus
Liberibacter psyllaurous.” This bacterium infects the psyllid
Bactericera cockerelli
and its solanaceous host plants potato and tomato, potentially resulting in “psyllid yellowing.” Host plant-dependent HLB transmission and variation in psyllid infection frequencies are found.
Enhancement of Recombinant Hemoglobin Production in
Escherichia coli
BL21(DE3) Containing the
Plesiomonas shigelloides
Heme Transport System
Authors: Villarreal D. M. , Phillips C. L. , Kelley A. M. , Villarreal S. , Villaloboz A. , Hernandez P. , Olson J. S. , Henderson D. P.
Published: August 2008

To produce recombinant hemoglobin in
Escherichia coli
, sufficient intracellular heme must be present, or the protein folds improperly and is degraded. In this study, coexpression of human hemoglobin genes and
Plesiomonas shigelloides
heme transport genes enhanced recombinant hemoglobin production in
E. coli
BL21(DE3) grown in medium containing heme.
Molecular Detection of Fungal Communities in the Hawaiian Marine Sponges
Suberites zeteki
and
Mycale armata
Authors: Gao Zheng , Li Binglin , Zheng Chengchao , Wang Guangyi
Published: August 2008

Symbiotic microbes play a variety of fundamental roles in the health and habitat ranges of their hosts. While prokaryotes in marine sponges have been broadly characterized, the diversity of sponge-inhabiting fungi has barely been explored using molecular approaches. Fungi are an important component of many marine and terrestrial ecosystems, and they may be an ecologically significant group in sponge-microbe interactions. This study tested the feasibility of using existing fungal primers for molecular analysis of sponge-associated fungal communities. None of the eight selected primer pairs yielded satisfactory results in fungal rRNA gene or internal transcribed spacer (ITS) clone library constructions. However, 3 of 10 denaturing gradient gel electrophoresis (DGGE) primer sets, which were designed to preferentially amplify fungal rRNA gene or ITS regions from terrestrial environmental samples, were successfully amplified from fungal targets in marine sponges. DGGE analysis indicated that fungal communities differ among different sponge species (
Suberites zeteki
and
Mycale armata
) and also vary between sponges and seawater. Sequence analysis of DGGE bands identified 23 and 21 fungal species from each of the two sponge species
S. zeteki
and
M. armata
, respectively. These species were representatives of 11 taxonomic orders and belonged to the phyla of
Ascomycota
(seven orders) and
Basidiomycota
(four orders). Five of these taxonomic orders (
Malasseziales, Corticiales, Polyporales, Agaricales
, and
Dothideomycetes et Chaetothyriomcetes incertae sedis
) have now been identified for the first time in marine sponges. Seven and six fungal species from
S. zeteki
and
M. armata
, respectively, are potentially new species because of their low sequence identity (≤98%) with their references in GenBank. Phylogenetic analysis indicated sponge-derived sequences were clustered into “marine fungus clades” with those from other marine habitats. This is the first report of molecular analysis of fungal communities in marine sponges, adding depth and dimension to our understanding of sponge-associated microbial communities.
Genetic Diversity of Native Bradyrhizobia Isolated from Soybeans (Glycine maxL.) in Different Agricultural-Ecological-Climatic Regions of India
Authors: Appunu Chinnaswamy , N′Zoue Angèle , Laguerre Gisèle
Published: August 2008

Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and thenifHandnodCgenes. Eight haplotypes assigned to theBradyrhizobiumgenus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and thednaK, glnII, recA, andnifHgenes revealed three groups. One of them (26% of isolates) was assigned toBradyrhizobium liaoningense. A second group (36% of isolates) was identified asB. yuanmingensebut likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all describedBradyrhizobiumspecies but showed the same symbiotic genotype asB. liaoningenseandB. japonicumbv.glycinearum.
Characterization of a Serine Proteinase Mediating Encystation of
Acanthamoeba
Authors: Moon Eun-Kyung , Chung Dong-Il , Hong Yeon-Chul , Kong Hyun-Hee
Published: August 2008

Members of the genus
Acanthamoeba
, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in
Acanthamoeba
, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of
Acanthamoeba
was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of
Acanthamoeba
. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of
Acanthamoeba
. An EMSP-enhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of
Acanthamoeba
by promoting autolysis.
Sorting Signals within the
Saccharomyces cerevisiae
Sporulation-Specific Dityrosine Transporter, Dtr1p, C Terminus Promote Golgi-to-Prospore Membrane Transport
Authors: Morishita Masayo , Engebrecht JoAnne
Published: August 2008

During sporulation in
Saccharomyces cerevisiae
, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a
cis
-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a
trans
-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.
Involvement of Sib Proteins in the Regulation of Cellular Adhesion in
Dictyostelium discoideum
Authors: Cornillon Sophie , Froquet Romain , Cosson Pierre
Published: August 2008

Molecular mechanisms ensuring cellular adhesion have been studied in detail in
Dictyostelium
amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in
Dictyostelium
, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a
Dictyostelium
population. In order to understand the mechanism underlying this regulation, we analyzed the expression of recently identified
Dictyostelium
adhesion molecules (Sib proteins) that present features also found in mammalian integrins.
sibA
and
sibC
are both expressed in vegetative
Dictyostelium
cells, but the expression of
sibC
is repressed strongly in conditions where cellular adhesion decreases. Analysis of
sibA
and
sibC
mutant cells further suggests that variations in the expression levels of
sibC
account largely for changes in cellular adhesion in response to environmental cues.
New Biosynthetic Step in the Melanin Pathway ofWangiella(Exophiala)dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor
Authors: Wheeler Michael H. , Abramczyk Dariusz , Puckhaber Lorraine S. , Naruse Michinori , Ebizuka Yutaka , Fujii Isao , Szaniszlo Paul J.
Published: August 2008

The predominant cell wall melanin ofWangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment inAspergillus fumigatusis produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant ofW. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albinoW. dermatitidisstrain with an inactivated WdPKS1gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN inW. dermatitidis.
Nucleus-Specific Importin Alpha Proteins and Nucleoporins Regulate Protein Import and Nuclear Division in the Binucleate
Tetrahymena thermophila
Authors: Malone Colin D. , Falkowska Katarzyna A. , Li Alanna Y. , Galanti Sarah E. , Kanuru Reshi C. , LaMont Elizabeth G. , Mazzarella Kate C. , Micev Alan J. , Osman Morwan M. , Piotrowski Nicholas K. , Suszko Jason W. , Timm Adam C. , Xu Ming-Ming , Liu Lucy , Chalker Douglas L.
Published: August 2008

The ciliate
Tetrahymena thermophila
, having both germ line micronuclei and somatic macronuclei, must possess a specialized nucleocytoplasmic transport system to import proteins into the correct nucleus. To understand how
Tetrahymena
can target proteins to distinct nuclei, we first characterized FG repeat-containing nucleoporins and found that micro- and macronuclei utilize unique subsets of these proteins. This finding implicates these proteins in the differential permeability of the two nuclei and implies that nuclear pores with discrete specificities are assembled within a single cell. To identify the import machineries that interact with these different pores, we characterized the large families of karyopherin homologs encoded within the genome. Localization studies of 13 putative importin (imp) α- and 11 imp β-like proteins revealed that imp α-like proteins are nucleus specific—nine localized to the germ line micronucleus—but that most imp β-like proteins localized to both types of nuclei. These data suggest that micronucleus-specific proteins are transported by specific imp α adapters. The different imp α proteins exhibit substantial sequence divergence and do not appear to be simply redundant in function. Disruption of the
IMA10
gene encoding an imp α-like protein that accumulates in dividing micronuclei results in nuclear division defects and lethality. Thus, nucleus-specific protein import and nuclear function in
Tetrahymena
are regulated by diverse, specialized karyopherins.
Dissecting Colony Development ofNeurospora crassaUsing mRNA Profiling and Comparative Genomics Approaches
Authors: Kasuga Takao , Glass N. Louise
Published: August 2008

Colony development, which includes hyphal extension, branching, anastomosis, and asexual sporulation, is a fundamental aspect of the life cycle of filamentous fungi; genetic mechanisms underlying these phenomena are poorly understood. We conducted transcriptional profiling during colony development of the model filamentous fungusNeurospora crassa, using 70-mer oligonucleotide microarrays. Relative mRNA expression levels were determined for six sections of defined age excised from a 27-h-oldN. crassacolony. Functional category analysis showed that the expression of genes involved in cell membrane biosynthesis, polar growth, and cellular signaling was enriched at the periphery of the colony. The relative expression of genes involved in protein synthesis and energy production was enriched in the middle section of the colony, while sections of the colony undergoing asexual development (conidiogenesis) were enriched in expression of genes involved in protein/peptide degradation and unclassified proteins. A cross-examination of theN. crassadata set with a published data set ofAspergillus nigerrevealed shared patterns in the spatiotemporal regulation of gene orthologs during colony development. At present, less than 50% of genes inN. crassahave functional annotation, which imposes the chief limitation on data analysis. Using an evolutionary approach, we observed that the expression of phylogenetically conserved groups of genes was enriched in the middle section of anN. crassacolony whereas expression of genes unique to euascomycete species and ofN. crassaorphan genes was enriched at the colony periphery and in the older, conidiating sections of a fungal colony.
Protein Import into Hydrogenosomes ofTrichomonas vaginalisInvolves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases
Authors: Mentel Marek , Zimorski Verena , Haferkamp Patrick , Martin William , Henze Katrin
Published: August 2008

The parabasalian flagellateTrichomonas vaginalisharbors mitochondrion-related and H2-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however,T. vaginalisis regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfectedT. vaginaliscells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.
A Protein with Similarity to PTEN Regulates Aggregation Territory Size by Decreasing Cyclic AMP Pulse Size during
Dictyostelium discoideum
Development
Authors: Tang Yitai , Gomer Richard H.
Published: August 2008

An interesting but largely unanswered biological question is how eukaryotic organisms regulate the size of multicellular tissues. During development, a lawn of
Dictyostelium
cells breaks up into territories, and within the territories the cells aggregate in dendritic streams to form groups of ∼20,000 cells. Using random insertional mutagenesis to search for genes involved in group size regulation, we found that an insertion in the
cnrN
gene affects group size. Cells lacking CnrN (
cnrN
−
) form abnormally small groups, which can be rescued by the expression of exogenous CnrN. Relayed pulses of extracellular cyclic AMP (cAMP) direct cells to aggregate by chemotaxis to form aggregation territories and streams.
cnrN
−
cells overaccumulate cAMP during development and form small territories. Decreasing the cAMP pulse size by treating
cnrN
−
cells with cAMP phosphodiesterase or starving
cnrN
−
cells at a low density rescues the small-territory phenotype. The predicted CnrN sequence has similarity to phosphatase and tensin homolog (PTEN), which in
Dictyostelium
inhibits cAMP-stimulated phosphatidylinositol 3-kinase signaling pathways. CnrN inhibits cAMP-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation, Akt activation, actin polymerization, and cAMP production. Our results suggest that CnrN is a protein with some similarities to PTEN and that it regulates cAMP signal transduction to regulate territory size.