bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alignment of the
c
-Type Cytochrome OmcS along Pili of
Geobacter sulfurreducens
Authors: Leang Ching , Qian Xinlei , Mester Tünde , Lovley Derek R. 
Published: April 2010

ABSTRACT

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by
Geobacter sulfurreducens
, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.
Purification and Characterization of OmcZ, an Outer-Surface, Octaheme
c
-Type Cytochrome Essential for Optimal Current Production by
Geobacter sulfurreducens
Authors: Inoue Kengo , Qian Xinlei , Morgado Leonor , Kim Byoung-Chan , Mester Tünde , Izallalen Mounir , Salgueiro Carlos A. , Lovley Derek R. 
Published: April 2010

ABSTRACT

Previous studies have demonstrated that
Geobacter sulfurreducens
requires the
c
-type cytochrome OmcZ, which is present in large (OmcZ
L
; 50-kDa) and small (OmcZ
S
; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZ
S
was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZ
S
and molecular weight measurements indicated that OmcZ
S
is a cleaved product of OmcZ
L
retaining all 8 hemes, including 1 heme with the unusual
c
-type heme-binding motif CX
14
CH. The purified OmcZ
S
was remarkably thermally stable (thermal-denaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZ
S
is approximately −220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (−420 to −60 mV). OmcZ
S
transferred electrons
in vitro
to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of
G. sulfurreducens
.
Comparative Transcriptome and Secretome Analysis of Wood Decay FungiPostia placentaandPhanerochaete chrysosporium
Authors: Vanden Wymelenberg Amber , Gaskell Jill , Mozuch Michael , Sabat Grzegorz , Ralph John , Skyba Oleksandr , Mansfield Shawn D. , Blanchette Robert A. , Martinez Diego , Grigoriev Igor , Kersten Philip J. , Cullen Dan 
Published: April 2010

ABSTRACTCellulose degradation by brown rot fungi, such asPostia placenta, is poorly understood relative to the phylogenetically related white rot basidiomycete,Phanerochaete chrysosporium. To elucidate the number, structure, and regulation of genes involved in lignocellulosic cell wall attack, secretome and transcriptome analyses were performed on both wood decay fungi cultured for 5 days in media containing ball-milled aspen or glucose as the sole carbon source. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a total of 67 and 79 proteins were identified in the extracellular fluids ofP. placentaandP. chrysosporiumcultures, respectively. Viewed together with transcript profiles,P. chrysosporiumemploys an array of extracellular glycosyl hydrolases to simultaneously attack cellulose and hemicelluloses. In contrast, under these same conditions,P. placentasecretes an array of hemicellulases but few potential cellulases. The two species display distinct expression patterns for oxidoreductase-encoding genes. InP. placenta, these patterns are consistent with an extracellular Fenton system and include the upregulation of genes involved in iron acquisition, in the synthesis of low-molecular-weight quinones, and possibly in redox cycling reactions.
Expression and Function of Four Carbonic Anhydrase Homologs in the Deep-Sea ChemolithoautotrophThiomicrospira crunogena
Authors: Dobrinski Kimberly P. , Boller Amanda J. , Scott Kathleen M. 
Published: April 2010

ABSTRACTThe hydrothermal vent chemolithoautotrophThiomicrospira crunogenagrows rapidly in the presence of low concentrations of dissolved inorganic carbon (DIC) (= CO2+ HCO3−+ CO3−2). Its genome encodes α-carbonic anhydrase (α-CA), β-CA, carboxysomal β-like CA (CsoSCA), and a protein distantly related to γ-CA. The purposes of this work were to characterize the gene products, determine whether they were differentially expressed, and identify those that are necessary for DIC uptake and fixation. When expressed inEscherichia coli, CA activity was detectable for α-CA, β-CA, and CsoSCA but not for the γ-CA-like protein. α-CA and CsoSCA but not β-CA were inhibited by sulfonamide inhibitors. CsoSCA was also inhibited by dithiothreitol. When grown under DIC limitation in chemostats,T. crunogenatranscribedcsoSCAmore frequently than when ammonia limited, while genes encoding α-CA and β-CA were not differentially transcribed under these conditions. Cell extracts fromT. crunogenagrown under both DIC- and ammonia-limited conditions had CA activity that was strongly inhibited by sulfonamides, though extracts from nitrogen-limited cells had some CA activity that was resistant, perhaps due to a higher level of β-CA activity. Based on predictions from the SignalP software program, subcellular location when expressed inE. coli, and carbonic anhydrase assays conducted on intactT. crunogenacells, α-CA is located in the periplasm. However, inhibition of α-CA by acetazolamide had only a minor impact on rates of DIC uptake or fixation. Conversely, inhibition of CsoSCA with ethoxyzolamide inhibited carbon fixation but not DIC uptake, consistent with this enzyme functioning to facilitate DIC interconversion and fixation within carboxysomes.
Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes
Authors: Moraïs Sarah , Barak Yoav , Caspi Jonathan , Hadar Yitzhak , Lamed Raphael , Shoham Yuval , Wilson David B. , Bayer Edward A. 
Published: April 2010

ABSTRACT

Conversion of components of the
Thermobifida fusca
free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the
T. fusca
xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ∼1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.
Production of Chiral (
R
)-3-Hydroxyoctanoic Acid Monomers, Catalyzed by
Pseudomonas fluorescens
GK13 Poly(3-Hydroxyoctanoic Acid) Depolymerase
Authors: Gangoiti Joana , Santos Marta , Llama María J. , Serra Juan L. 
Published: April 2010

ABSTRACT

The extracellular medium-chain-length polyhydroxyalkanoate (MCL-PHA) depolymerase of
Pseudomonas fluorescens
GK13 catalyzes the hydrolysis of poly(3-hydroxyoctanoic acid) [P(3HO)]. Based on the strong tendency of the enzyme to interact with hydrophobic materials, a low-cost method which allows the rapid and easy purification and immobilization of the enzyme has been developed. Thus, the extracellular P(3HO) depolymerase present in the culture broth of cells of
P. fluorescens
GK13 grown on mineral medium supplemented with P(3HO) as the sole carbon and energy source has been tightly adsorbed onto a commercially available polypropylene support (Accurel MP-1000) with high yield and specificity. The activity of the pure enzyme was enhanced by the presence of detergents and organic solvents, and it was retained after treatment with an SDS-denaturing cocktail under both reducing and nonreducing conditions. The time course of the P(3HO) hydrolysis catalyzed by the soluble and immobilized enzyme has been assessed, and the resulting products have been identified. After 24 h of hydrolysis, the dimeric ester of 3-HO [(
R
)-3-HO-HO] was obtained as the main product of the soluble enzyme. However, the immobilized enzyme catalyzes almost the complete hydrolysis of P(3HO) polymer to (
R
)-3-HO monomers under the same conditions.
Primary Gut Symbiont and Secondary,
Sodalis
-Allied Symbiont of the Scutellerid Stinkbug
Cantao ocellatus
Authors: Kaiwa Nahomi , Hosokawa Takahiro , Kikuchi Yoshitomo , Nikoh Naruo , Meng Xian Ying , Kimura Nobutada , Ito Motomi , Fukatsu Takema 
Published: April 2010

ABSTRACT

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of
Cantao ocellatus
, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the
Gammaproteobacteria
, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and
in situ
hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of
C. ocellatus
harbored another bacterial symbiont in their gonads, which was closely related to
Sodalis glossinidius
, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary
Sodalis
-allied symbiont are discussed.
Development of a Chip Assay and Quantitative PCR for Detecting Microcystin Synthetase E Gene Expression
Authors: Sipari Hanna , Rantala-Ylinen Anne , Jokela Jouni , Oksanen Ilona , Sivonen Kaarina 
Published: April 2010

ABSTRACT

The chip and quantitative real-time PCR (qPCR) assays were optimized to study the expression of microcystin biosynthesis genes (
mcy
) with RNA samples extracted from cyanobacterial strains and environmental water samples. Both microcystin-producing
Anabaena
and
Microcystis
were identified in Lake Tuusulanjärvi samples.
Microcystis
transcribed the
mcyE
genes throughout the summer of 2006, while expression by
Anabaena
became evident later in August and September. Active
mcyE
gene expression was also detectable when microcystin concentrations were very low. Detection of
Anabaena mcyE
transcripts by qPCR, as well as certain cyanobacterial 16S rRNAs with the chip assay, showed slightly reduced sensitivity compared with the DNA analyses. In contrast, even groups undetectable or present in low quantities as determined by microscopy could be identified with the chip assay from DNA samples. The methods introduced add to the previously scarce repertoire of applications for
mcy
expression profiling in environmental samples and enable
in situ
studies of regulation of microcystin synthesis in response to environmental factors.
Monomethylamine as a Nitrogen Source for a Nonmethylotrophic Bacterium,
Agrobacterium tumefaciens
Authors: Chen Yin , McAleer Kathryn L. , Murrell J. Colin 
Published: April 2010

ABSTRACT

Monomethylamine can be used by nonmethylotrophs as a sole nitrogen source but not as a carbon source; however, little is known about the genes and enzymes involved. The γ-glutamylmethylamide/
N
-methylglutamate pathway for monomethylamine utilization by methylotrophs has recently been resolved. We have identified genes encoding key enzymes of this pathway in nonmethylotrophs (e.g.,
Agrobacterium tumefaciens
) and demonstrated that this pathway is also involved in the utilization of monomethylamine as a nitrogen source by nonmethylotrophs.
Bioluminescence Imaging ofClavibacter michiganensissubsp.michiganensisInfection of Tomato Seeds and Plants
Authors: Xu Xiulan , Miller Sally A. , Baysal-Gurel Fulya , Gartemann Karl-Heinz , Eichenlaub Rudolf , Rajashekara Gireesh 
Published: April 2010

ABSTRACTClavibacter michiganensissubsp.michiganensisis a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement ofC. michiganensissubsp.michiganensisin grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on theC. michiganensissubsp.michiganensismovement in tomato seed and grafted plants, we isolated a bioluminescentC. michiganensissubsp.michiganensisstrain using the modified Tn1409containing a promoterlessluxreporter. A total of 19 bioluminescentC. michiganensissubsp.michiganensismutants were obtained. All mutants tested induced a hypersensitive response inMirabilis jalapaand caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed thatC. michiganensissubsp.michiganensisaggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescentC. michiganensissubsp.michiganensisto help better elucidate theC. michiganensissubsp.michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation ofC. michiganensissubsp.nebraskensiswith Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally importantC. michiganensis.
Relationship between Virulence Gene Profiles of Atypical Enteropathogenic
Escherichia coli
and Shiga Toxin- Producing
E. coli
Isolates from Cattle and Sheep in New Zealand
Authors: Cookson Adrian L. , Cao Mingshu , Bennett Jenny , Nicol Carolyn , Thomson-Carter Fiona , Attwood Graeme T. 
Published: April 2010

ABSTRACT

Virulence gene profiles of atypical enteropathogenic
Escherichia coli
(aEPEC) and Shiga toxin-producing
E. coli
(STEC) from cattle, sheep, and humans were examined to determine the relationship between pathotypes. Shared virulence factors (intimin, EHEC hemolysin, serine protease, and a type II secretion system) were identified, suggesting a dynamic evolutionary relationship between aEPEC and STEC.
Identification and Typing of
Lactococcus lactis
by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Authors: Tanigawa Kana , Kawabata Hiroaki , Watanabe Koichi 
Published: April 2010

ABSTRACT

Currently, the genus
Lactococcus
is classified into six species:
Lactococcus chungangensis
,
L. garvieae
,
L. lactis
,
L. piscium
,
L. plantarum
, and
L. raffinolactis
. Among these six species,
L. lactis
is especially important because of its use in the manufacture of probiotic dairy products.
L. lactis
consists of three subspecies:
L. lactis
subsp.
cremoris
,
L. lactis
subsp.
hordniae
, and
L. lactis
subsp.
lactis
. However, these subspecies have not yet been reliably discriminated. To date, mainly phenotypic identification has been used, with a few genotypic identifications. We discriminated species or subspecies in the genus
Lactococcus
not only by proteomics identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) but also by phenotypic and genotypic identification. The proteomics identification using differences in the mass spectra of ribosomal proteins was nearly identical to that by genotypic identification (i.e., by analyses of 16S rRNA and
recA
gene sequences and amplified fragment length polymorphism). The three ribosomal subunits 30S/L31, 50S/L31, and 50S/L35 were the best markers for discriminating
L. lactis
subsp.
cremoris
from
L. lactis
subsp.
lactis
. Proteomics identification using MALDI-TOF MS was therefore a powerful method for discriminating and identifying these bacteria. In addition, this method was faster and more reliable than others that we examined.
Characterization of Genes Responsible for the CO-Linked Hydrogen Production Pathway in
Rubrivivax gelatinosus
Authors: Vanzin Gary , Yu Jianping , Smolinski Sharon , Tek Vekalet , Pennington Grant , Maness Pin-Ching 
Published: April 2010

ABSTRACT

Upon exposure to carbon monoxide, the purple nonsulfur photosynthetic bacterium
Rubrivivax gelatinosus
produces hydrogen concomitantly with the oxidation of CO according to the equation CO + H
2
O ↔ CO
2
+ H
2
. Yet little is known about the genetic elements encoding this reaction in this organism. In the present study, we use transposon mutagenesis and functional complementation to uncover three clustered genes,
cooL
,
cooX
, and
cooH
, in
Rubrivivax gelatinosus
putatively encoding part of a membrane-bound, multisubunit NiFe-hydrogenase. We present the complete amino acid sequences for the large catalytic subunit and its electron-relaying small subunit, encoded by
cooH
and
cooL
, respectively. Sequence alignment reveals a conserved region in the large subunit coordinating a binuclear [NiFe] center and a conserved region in the small subunit coordinating a [4Fe-4S] cluster. Protein purification experiments show that a protein fraction of 58 kDa molecular mass could function in H
2
evolution mediated by reduced methyl viologen. Western blotting experiments show that the two hydrogenase subunits are detectable and accumulate only when cells are exposed to CO. The
cooX
gene encodes a putative Fe-S protein mediating electron transfer to the hydrogenase small subunit. We conclude that these three
Rubrivivax
proteins encompass part of a membrane-bound, multisubunit NiFe-hydrogenase belonging to the energy-converting hydrogenase (Ech) type, which has been found among diverse microbes with a common feature in coupling H
2
production with proton pumping for energy generation.
Antibiotic Resistance Characteristics of Environmental Bacteria from an Oxytetracycline Production Wastewater Treatment Plant and the Receiving River
Authors: Li Dong , Yu Tao , Zhang Yu , Yang Min , Li Zhen , Liu Miaomiao , Qi Rong 
Published: April 2010

ABSTRACT

We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as
Gammaproteobacteria
. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (
tet
) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with
tet
(A) being the most common (67.0%), followed by
tet
(W),
tet
(C),
tet
(J),
tet
(L),
tet
(D),
tet
(Y), and
tet
(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the
tet
genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.
Genetic Tool Development for a New Host for Biotechnology, the Thermotolerant Bacterium
Bacillus coagulans
Authors: Kovács Ákos T. , van Hartskamp Mariska , Kuipers Oscar P. , van Kranenburg Richard 
Published: April 2010

ABSTRACT

Bacillus coagulans
has good potential as an industrial production organism for platform chemicals from renewable resources but has limited genetic tools available. Here, we present a targeted gene disruption system using the Cre-
lox
system, development of a LacZ reporter assay for monitoring gene transcription, and heterologous
d
-lactate dehydrogenase expression.