bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Assembly of Minicellulosomes on the
Saccharomyces cerevisiae
Cell Surface for Cellulose Hydrolysis and Ethanol Production
Authors: Tsai Shen-Long , Oh Jeongseok , Singh Shailendra , Chen Ruizhen , Chen Wilfred 
Published: August 2009

ABSTRACT

We demonstrated the functional display of a miniscaffoldin on the
Saccharomyces cerevisiae
cell surface consisting of three divergent cohesin domains from
Clostridium thermocellum
(t),
Clostridium cellulolyticum
(c), and
Ruminococcus flavefaciens
(f). Incubation with
Escherichia coli
lysates containing an endoglucanase (CelA) fused with a dockerin domain from
C. thermocellum
(At), an exoglucanase (CelE) from
C. cellulolyticum
fused with a dockerin domain from the same species (Ec), and an endoglucanase (CelG) from
C. cellulolyticum
fused with a dockerin domain from
R. flavefaciens
(Gf) resulted in the assembly of a functional minicellulosome on the yeast cell surface. The displayed minicellulosome retained the synergistic effect for cellulose hydrolysis. When a β-glucosidase (BglA) from
C. thermocellum
tagged with the dockerin from
R. flavefaciens
was used in place of Gf, cells displaying the new minicellulosome exhibited significantly enhanced glucose liberation and produced ethanol directly from phosphoric acid-swollen cellulose. The final ethanol concentration of 3.5 g/liter was 2.6-fold higher than that obtained by using the same amounts of added purified cellulases. The overall yield was 0.49 g of ethanol produced per g of carbohydrate consumed, which corresponds to 95% of the theoretical value. This result confirms that simultaneous and synergistic saccharification and fermentation of cellulose to ethanol can be efficiently accomplished with a yeast strain displaying a functional minicellulosome containing all three required cellulolytic enzymes.
Inducible-Expression Plasmid for
Rhodobacter sphaeroides
and
Paracoccus denitrificans
Authors: Ind Alice C. , Porter Steven L. , Brown Mostyn T. , Byles Elaine D. , de Beyer Jennifer A. , Godfrey Scott A. , Armitage Judith P. 
Published: August 2009

ABSTRACT

We have developed a stable isopropyl-β-
d
-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria
Rhodobacter sphaeroides
and
Paracoccus denitrificans
. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the
lacI
q
gene from pYanni3 and the
lac
promoter, P
A1/04/03
, from pJBA24.
Independent Transport and Sorting of Functionally Distinct Protein Families in
Tetrahymena thermophila
Dense Core Secretory Granules
Authors: Rahaman Abdur , Miao Wei , Turkewitz Aaron P. 
Published: August 2009

ABSTRACT

Dense core granules (DCGs) in
Tetrahymena thermophila
contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (
gr
anule
t
ip), which was previously identified as an abundant protein in this family. Cells lacking all copies of
GRT1
, together with the closely related
GRT2
, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major
GRL
genes, Δ
GRT1
Δ
GRT2
cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on
GRT1
. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Δ
GRT1
Δ
GRT2
cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in
Tetrahymena
DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Δ
GRT1
Δ
GRT2
cells appear less adhesive than those from the wild type.
Identification of
Francisella tularensis
Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species
Authors: Melillo Amanda A. , Mahawar Manish , Sellati Timothy J. , Malik Meenakshi , Metzger Dennis W. , Melendez J. Andres , Bakshi Chandra Shekhar 
Published: August 2009

ABSTRACT

Francisella tularensis
is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O
2
−
). In the present study, mutants of
F
.
tularensis
live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion
F
.
tularensis
mutant of
sodC
(Δ
sodC
) and a
F
.
tularensis
Δ
sodC
mutant with attenuated Fe-superoxide dismutase (
sodB
) gene expression (
sodB
Δ
sodC
) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The
F
.
tularensis
Δ
sodC
and
sodB
Δ
sodC
mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type
F
.
tularensis
LVS. Transcomplementing the
sodC
gene in the Δ
sodC
mutant or inhibiting the IFN-γ-dependent production of O
2
−
or nitric oxide (NO) enhanced intramacrophage survival of the
sod
mutants. The Δ
sodC
and
sodB
Δ
sodC
mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type
F
.
tularensis
LVS. As observed for macrophages, the virulence of the Δ
sodC
mutant was restored in
ifn
-γ
−/−
,
inos
−
/
−
, and
phox
−
/
−
mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of
F
.
tularensis
in mice.
Structural and Mutational Analysis of Band 7 Proteins in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Boehm Marko , Nield Jon , Zhang Pengpeng , Aro Eva-Mari , Komenda Josef , Nixon Peter J. 
Published: August 2009

ABSTRACT

Band 7 proteins, which encompass members of the stomatin, prohibitin, flotillin, and HflK/C protein families, are integral membrane proteins that play important physiological roles in eukaryotes but are poorly characterized in bacteria. We have studied the band 7 proteins encoded by the cyanobacterium
Synechocystis
sp. strain PCC 6803, with emphasis on their structure and proposed role in the assembly and maintenance of the photosynthetic apparatus. Mutagenesis revealed that none of the five band 7 proteins (Slr1106, Slr1128, Slr1768, Sll0815, and Sll1021) was essential for growth under a range of conditions (including high light, salt, oxidative, and temperature stresses), although motility was compromised in an Slr1768 inactivation mutant. Accumulation of the major photosynthetic complexes in the thylakoid membrane and repair of the photosystem II complex following light damage were similar in the wild type and a quadruple mutant. Cellular fractionation experiments indicated that three of the band 7 proteins (Slr1106, Slr1768, and Slr1128) were associated with the cytoplasmic membrane, whereas Slr1106, a prohibitin homologue, was also found in the thylakoid membrane fraction. Blue native gel electrophoresis indicated that these three proteins, plus Sll0815, formed large (>669-kDa) independent complexes. Slr1128, a stomatin homologue, has a ring-like structure with an approximate diameter of 16 nm when visualized by negative stain electron microscopy. No evidence for band 7/FtsH supercomplexes was found. Overall, our results indicate that the band 7 proteins form large homo-oligomeric complexes but do not play a crucial role in the biogenesis of the photosynthetic apparatus in
Synechocystis
sp. strain PCC 6803.
Site-Specific Insertion of IS
492
in
Pseudoalteromonas atlantica
Authors: Higgins Brian P. , Popkowski Adam C. , Caruana Peter R. , Karls Anna C. 
Published: August 2009

ABSTRACT

Reversible insertion of IS
492
at a site within
epsG
on the
Pseudoalteromonas atlantica
chromosome controls peripheral extracellular polysaccharide production and biofilm formation by
P. atlantica
. High-frequency precise excision of IS
492
from
epsG
requires 5 and 7 bp of flanking DNA, suggesting that IS
492
transposition involves a site-specific recombination mechanism. The site specificity of IS
492
insertion was examined in
P. atlantica
and shown to be specific for a 7-bp target, 5′-CTTGTTA-3′. Characterization of numerous insertion events at the target site in
epsG
indicated that insertion is also orientation specific. The frequency of IS
492
insertion at the
epsG
target site (2.7 × 10
−7
/cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS
492
precise excision from the same site. Comparison of insertion sites for IS
492
and the highly related IS
Ptu2
from
Pseudoalteromonas tunicata
suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS
492
.
Inactivation of VicK Affects Acid Production and Acid Survival of
Streptococcus mutans
Authors: Senadheera D. , Krastel K. , Mair R. , Persadmehr A. , Abranches J. , Burne R. A. , Cvitkovitch Dennis G. 
Published: August 2009

ABSTRACT

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium
Streptococcus mutans
have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the
S. mutans
VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (
P
< 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of
S. mutans
(
P
< 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and
S. mutans
wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (
P
< 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of
S. mutans
, here we imply a novel role for VicK in regulating intracellular pH homeostasis in
S. mutans
.
Malonic Semialdehyde Reductase, Succinic Semialdehyde Reductase, and Succinyl-Coenzyme A Reductase from
Metallosphaera sedula
: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in
Sulfolobales
Authors: Kockelkorn Daniel , Fuchs Georg 
Published: August 2009

ABSTRACT

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO
2
fixation in various members of the
Crenarchaea
. In this cycle, as determined using
Metallosphaera sedula
, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from
M. sedula
and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the
Metallosphaera
genes were found in other members of the
Sulfolobales
. Only distantly related genes were found in the genomes of autotrophic marine
Crenarchaeota
that may use a similar cycle in autotrophic carbon fixation.
Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX, DedD, and RlpA) in
Escherichia coli
Cell Constriction
Authors: Gerding Matthew A. , Liu Bing , Bendezú Felipe O. , Hale Cynthia A. , Bernhardt Thomas G. , de Boer Piet A. J. 
Published: August 2009

ABSTRACT

Of the known essential division proteins in
Escherichia coli
, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (
E
FtsN) of at most 35 residues that is centered about helix H2 in the periplasm.
E
FtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (
S
FtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of
S
FtsN depended on the ability of cells to constrict and, thus, on the activity of
E
FtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional
E. coli
proteins of unknown function, DamX, DedD, and RlpA, as well as that of
Bacillus subtilis
CwlC, also accumulated sharply at constriction sites in
E. coli
, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.
A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters inShigella
Authors: Janakiraman Anuradha , Fixen Kathryn R. , Gray Andrew N. , Niki Hironori , Goldberg Marcia B. 
Published: August 2009

ABSTRACTAutotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, theShigellaautotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library ofEscherichia coliproteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitiveftsZallele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm ofE. coli. DnaK was also required for IcsA secretion at the pole inShigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A secondShigellaautotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.
A New Member of the
Escherichia coli fad
Regulon: Transcriptional Regulation of
fadM
(
ybaW
)
Authors: Feng Youjun , Cronan John E. 
Published: August 2009

ABSTRACT

Recently, Nie and coworkers (L. Nie, Y. Ren, A. Janakiraman, S. Smith, and H. Schulz, Biochemistry 47:9618-9626, 2008) reported a new
Escherichia coli
thioesterase encoded by the
ybaW
gene that cleaves the thioester bonds of inhibitory acyl-coenzyme A (CoA) by-products generated during β-oxidation of certain unsaturated fatty acids. These authors suggested that
ybaW
expression might be regulated by FadR, the repressor of the
fad
(fatty acid degradation) regulon. We report mapping of the
ybaW
promoter and show that
ybaW
transcription responded to FadR in vivo. Moreover, purified FadR bound to a DNA sequence similar to the canonical FadR binding site located upstream of the
ybaW
coding sequence and was released from the promoter upon the addition of long-chain acyl-CoA thioesters. We therefore propose the designation
fadM
in place of
ybaW
. Although FadR regulation of
fadM
expression had the pattern typical of
fad
regulon genes, its modulation by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) global regulator was the opposite of that normally observed. CRP-cAMP generally acts as an activator of
fad
gene expression, consistent with the low status of fatty acids as carbon sources. However, glucose growth stimulated
fadM
expression relative to acetate growth, as did inactivation of CRP-cAMP, indicating that the complex acts as a negative regulator of this gene. The stimulation of
fadM
expression seen upon deletion of the gene encoding adenylate cyclase (Δ
cya
) was reversed by supplementation of the growth medium with cAMP. Nie and coworkers also reported that growth on a conjugated linoleic acid isomer yields much higher levels of FadM thioesterase activity than does growth on oleic acid. In contrast, we found that the conjugated linoleic acid isomer was only a weak inducer of
fadM
expression. Although the gene is not essential for growth, the high basal level of
fadM
expression under diverse growth conditions suggests that the encoded thioesterase has functions in addition to β-oxidation.
The Iho670 Fibers of
Ignicoccus hospitalis
: a New Type of Archaeal Cell Surface Appendage
Authors: Müller Daniel W. , Meyer Carolin , Gürster Sonja , Küper Ulf , Huber Harald , Rachel Reinhard , Wanner Gerhard , Wirth Reinhard , Bellack Annett 
Published: August 2009

ABSTRACT

Ignicoccus hospitalis
forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 μm), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle.
The Multifaceted Proteins MvaT and MvaU, Members of the H-NS Family, Control Arginine Metabolism, Pyocyanin Synthesis, and Prophage Activation in
Pseudomonas aeruginosa
PAO1
Authors: Li Congran , Wally Hassan , Miller Shearman J. , Lu Chung-Dar 
Published: August 2009

ABSTRACT

The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the
aotJQMOP-argR
operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the
aotJ
regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in
mvaT
or
mvaU
, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the β-galactosidase activities from
aotJ
::
lacZ
fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of
aotJ
expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of
cupA
expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on
cupA
. Moreover, prophage Pf4 integrated into the chromosome of
Pseudomonas aeruginosa
PAO1 was activated in an
mvaT mvaU
double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the
mvaT mvaU
double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in
P. aeruginosa
.