bluuebunny/crossref_metadata_2025 huggingface dataset:
Observation of Reform Teaching in Undergraduate Level Mathematics and Science Courses
Authors: Wainwright Camille , Morrell Patricia D. , Flick Larry , Schepige Adele
Published: April 2010

This paper reports on initial results from an ongoing evaluation study of a National Science Foundation project to implement reform‐oriented teaching practices in college science and mathematics courses. The purpose of this study was to determine what elements of reform teaching are being utilized by college faculty members teaching undergraduate science and mathematics courses, including a qualitative estimate of the frequency with which they are used. Participating instructors attended summer institutes that modeled reform‐based practices and fostered reflection on current issues in science, mathematics, and technological literacy for K‐16 teaching, with an explicit emphasis on the importance of creating the best possible learning experience for prospective K‐12 science and mathematics teachers. Utilizing a unique classroom observation protocol (the Oregon‐Teacher Observation Protocol) and interviews, the authors (a) conclude that some reform‐oriented teaching strategies are evident in undergraduate mathematics and science instruction and (b) suggest areas in which additional support and feedback are needed in order for higher education faculty members to adopt reform‐based instructional methodology.
Geometrical Anisotropy in Biphase Particle Reinforced Composites
Authors: Ranganathan Shivakumar I. , Decuzzi Paolo , Wheeler Lewis T. , Ferrari Mauro
Published: April 2010

Particle shape plays a crucial role in the design of novel reinforced composites. We introduce the notion of a geometrical anisotropy index A to characterize the particle shape and establish its relationship with the effective elastic constants of biphase composite materials. Our analysis identifies three distinct regions of A: (i) By using ovoidal particles with small A, the effective stiffness scales linearly with A for a given volume fraction α; (ii) for intermediate values of A, the use of prolate particles yield better elastic properties; and (iii) for large A, the use of oblate particles result in higher effective stiffness. Interestingly, the transition from (ii) to (iii) occurs at a critical anisotropy Acr and is independent of α.
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

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

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

Cellulose 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

The 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

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

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

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

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

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

Clavibacter 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

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

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

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

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

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.
Intracellular Accumulation of High Levels of γ-Aminobutyrate by
Listeria monocytogenes
10403S in Response to Low pH: Uncoupling of γ-Aminobutyrate Synthesis from Efflux in a Chemically Defined Medium
Authors: Karatzas Kimon-Andreas G. , Brennan Orla , Heavin Sinéad , Morrissey John , O'Byrne Conor P.
Published: April 2010

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of
Listeria monocytogenes
at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular γ-aminobutyrate (GABA
i
). The glutamate is then decarboxylated to GABA
i
, a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when
L. monocytogenes
10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA
e
) in response to acidification. In addition, high levels of GABA
i
(>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of
gadD1T1
and
gadD3
occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA
i
in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA
i
. These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.
Cellulosilyticum ruminicola
, a Newly Described Rumen Bacterium That Possesses Redundant Fibrolytic-Protein-Encoding Genes and Degrades Lignocellulose with Multiple Carbohydrate- Borne Fibrolytic Enzymes
Authors: Cai Shichun , Li Jiabao , Hu Fen Ze , Zhang Kegui , Luo Yuanming , Janto Benjamin , Boissy Robert , Ehrlich Garth , Dong Xiuzhu
Published: April 2010

Cellulosilyticum ruminicola
H1 is a newly described bacterium isolated from yak (
Bos grunniens
) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of
C. ruminicola
H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.
Birefringence and DNA Condensation of Liquid Crystalline Chromosomes
Authors: Chow Man H. , Yan Kosmo T. H. , Bennett Michael J. , Wong Joseph T. Y.
Published: April 2010

DNA can self-assemble
in vitro
into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes—up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.
Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans, but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase
Authors: Brown Sarah M. , Upadhya Rajendra , Shoemaker James D. , Lodge Jennifer K.
Published: April 2010

The opportunistic intracellular fungal pathogenCryptococcus neoformansdepends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochromecperoxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP+-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion ofZWF1inC. neoformansdid not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion ofIDP1resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet theidp1Δ strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance.
Naegleria gruberi
De Novo
Basal Body Assembly Occurs via Stepwise Incorporation of Conserved Proteins
Authors: Fritz-Laylin Lillian K. , Assaf Zoe June , Chen Sean , Cande W. Zacheus
Published: April 2010

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella.
Naegleria gruberi
, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed,
Naegleria
rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton
de novo
, including two basal bodies and flagella. Here, we show that
Naegleria
has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, γ-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that
Naegleria
expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally γ-tubulin. The similarities between basal body assembly in
Naegleria
and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.
A Role for the Class A Penicillin-Binding Protein PonA2 in the Survival ofMycobacterium smegmatisunder Conditions of Nonreplication
Authors: Patru Maria-Magdalena , Pavelka Martin S.
Published: April 2010

Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genusMycobacteriumhave been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes ofMycobacterium smegmatisand other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain apenicillin-binding proteinandserine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that anM. smegmatisΔponA2mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a ΔponA3mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 whenponA3is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied byM. smegmatisand other soil-dwelling mycobacteria.
YneA, an SOS-Induced Inhibitor of Cell Division in
Bacillus subtilis
, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity
Authors: Mo Allison H. , Burkholder William F.
Published: April 2010

Cell viability depends on the stable transmission of genetic information to each successive generation. Therefore, in the event of intrinsic or extrinsic DNA damage, it is important that cell division be delayed until DNA repair has been completed. In
Bacillus subtilis
, this is accomplished in part by YneA, an inhibitor of division that is induced as part of the SOS response. We sought to gain insight into the mechanism by which YneA blocks cell division and the processes involved in shutting off YneA activity. Our data suggest that YneA is able to inhibit daughter cell separation as well as septum formation. YneA contains a LysM peptidoglycan binding domain and is predicted to be exported. We established that the YneA signal peptide is rapidly cleaved, resulting in secretion of YneA into the medium. Mutations within YneA affect both the rate of signal sequence cleavage and the activity of YneA. YneA does not stably associate with the cell wall and is rapidly degraded by extracellular proteases. Based on these results, we hypothesize that exported YneA is active prior to signal peptide cleavage and that proteolysis contributes to the inactivation of YneA. Finally, we identified mutations in the transmembrane segment of YneA that abolish the ability of YneA to inhibit cell division, while having little or no effect on YneA export or stability. These data suggest that protein-protein interactions mediated by the transmembrane region may be required for YneA activity.
N-Terminal Type III Secretion Signal of Enteropathogenic
Escherichia coli
Translocator Proteins
Authors: Munera Diana , Crepin Valerie F. , Marches Olivier , Frankel Gad
Published: April 2010

We report that the N terminus of the type III secretion system translocator proteins EspB, EspD, and EspA mediate protein secretion and translocation from wild-type enteropathogenic
Escherichia coli
and hypersecretion from
sepL
and
sepD
mutants. EspA containing the translocation signal of Map and Tir containing the secretion signal of EspA are biologically active.
Host-Interactive Genes in AmerindianHelicobacter pyloriDiverge from Their Old World Homologs and Mediate Inflammatory Responses
Authors: Mane S. P. , Dominguez-Bello M. G. , Blaser M. J. , Sobral B. W. , Hontecillas R. , Skoneczka J. , Mohapatra S. K. , Crasta O. R. , Evans C. , Modise T. , Shallom S. , Shukla M. , Varon C. , Mégraud F. , Maldonado-Contreras A. L. , Williams K. P. , Bassaganya-Riera J.
Published: April 2010

Helicobacter pyloriis the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults.H. pyloripopulations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of thecag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparativeH. pylorigenomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing AmerindianH. pylorias a particularly close sister group to East AsianH. pylori. In contrast, phylogenetic analysis of the host-interactive genesvacAandcagAshows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in thecagpathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225dcagarchitecture was fully functional via conserved elements, but the natural deletion of 13cagpathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.
Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of
Shewanella oneidensis
Strain MR-1
Authors: Hunt Kristopher A. , Flynn Jeffrey M. , Naranjo Belén , Shikhare Indraneel D. , Gralnick Jeffrey A.
Published: April 2010

It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that
Shewanella oneidensis
strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking
ackA
(SO2915) and
pta
(SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking
ackA
and
pta
were also shown to grow slowly using
N
-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on
N
-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although
S. oneidensis
requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an
ackA
deletion in
Shewanella
sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of
Shewanella
.
A Unique Group of Virus-Related, Genome-Integrating Elements Found Solely in the Bacterial Family
Thermaceae
and the Archaeal Family
Halobacteriaceae
Authors: Jalasvuori Matti , Pawlowski Alice , Bamford Jaana K. H.
Published: April 2010

Viruses SH1 and P23-77, infecting archaeal
Haloarcula
species and bacterial
Thermus
species, respectively, were recently designated to form a novel viral lineage. In this study, the lineage is expanded to archaeal
Halomicrobium
and bacterial
Meiothermus
species by analysis of five genome-integrated elements that share the core genes with these viruses.
The Rut Pathway for Pyrimidine Degradation: Novel Chemistry and Toxicity Problems
Authors: Kim Kwang-Seo , Pelton Jeffrey G. , Inwood William B. , Andersen Ulla , Kustu Sydney , Wemmer David E.
Published: April 2010

The Rut pathway is composed of seven proteins, all of which are required byEscherichia coliK-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of18O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA.In vitroRutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E.In vivowe postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.
The Phosphoenolpyruvate Phosphotransferase System Regulates
Vibrio cholerae
Biofilm Formation through Multiple Independent Pathways
Authors: Houot Laetitia , Chang Sarah , Pickering Bradley S. , Absalon Cedric , Watnick Paula I.
Published: April 2010

The bacterial phosphoenolpyruvate phosphotransferase system (PTS) is a highly conserved phosphotransfer cascade that participates in the transport and phosphorylation of selected carbohydrates and modulates many cellular functions in response to carbohydrate availability. It plays a role in the virulence of many bacterial pathogens. Components of the carbohydrate-specific PTS include the general cytoplasmic components enzyme I (EI) and histidine protein (HPr), the sugar-specific cytoplasmic components enzymes IIA (EIIA) and IIB (EIIB), and the sugar-specific membrane-associated multisubunit components enzymes IIC (EIIC) and IID (EIID). Many bacterial genomes also encode a parallel PTS pathway that includes the EI homolog EI
Ntr
, the HPr homolog NPr, and the EIIA homolog EIIA
Ntr
. This pathway is thought to be nitrogen specific because of the proximity of the genes encoding this pathway to the genes encoding the nitrogen-specific σ factor σ
54
. We previously reported that phosphorylation of HPr and FPr by EI represses
Vibrio cholerae
biofilm formation in minimal medium supplemented with glucose or pyruvate. Here we report two additional PTS-based biofilm regulatory pathways that are active in LB broth but not in minimal medium. These pathways involve the glucose-specific enzyme EIIA (EIIA
Glc
) and two nitrogen-specific EIIA homologs, EIIA
Ntr1
and EIIA
Ntr2
. The presence of multiple, independent biofilm regulatory circuits in the PTS supports the hypothesis that the PTS and PTS-dependent substrates have a central role in sensing environments suitable for a surface-associated existence.
Identification of the Quorum-Sensing Target DNA Sequence andN-Acyl Homoserine Lactone Responsiveness of theBrucella abortus virBpromoter
Authors: Arocena Gastón M. , Sieira Rodrigo , Comerci Diego J. , Ugalde Rodolfo A.
Published: April 2010

VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogenBrucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including thevirBoperon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis ofBrucella. Although the regulatory role of VjbR on thevirBpromoter (PvirB) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in PvirBby DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR ofMesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in PvirBincreased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator ofBrucellais highly related to that ofM. tianshanense. The addition ofN-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of PvirB. Our results provide new molecular evidence for the structure of thevirBpromoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence inBrucella.