bluuebunny/crossref_metadata_2025_split huggingface dataset:
Denaturing Gradient Gel Electrophoresis as a Tool To Determine Batch Similarity of Probiotic Cultures of Porcine Cecal Bacteria
Authors: Hume Michael E. , Scanlan Charles M. , Harvey Roger B. , Andrews Kathleen , Snodgrass James D. , Nalian Armen G. , Martynova-Van Kley Alexandra , Nisbet David J. 
Published: June 2008

ABSTRACTA continuous-flow porcine cecal bacterial culture has been used experimentally as treatment against enterotoxigenicEscherichia coliin weanling pigs. Periodically, the cultures must be started from frozen stock. Our results indicate that denaturing gradient gel electrophoresis can be applied as an indirect indication of culture similarity for each new batch generated from frozen stock.
Isolation of Surfactant-Resistant Bacteria from Natural, Surfactant-Rich Marine Habitats
Authors: Plante Craig J. , Coe Kieran M. , Plante Rebecca G. 
Published: June 2008

ABSTRACTEnvironmental remediation efforts often utilize either biodegradative microbes or surfactants, but not in combination. Coupling both strategies holds the potential to dramatically increase the rate and extent of remediation because surfactants can enhance the bioavailability of contaminants to microbes. However, many surfactants permeabilize bacterial cell membranes and are effective disinfectants. An important goal then is to find or genetically modify microorganisms that possess both desirable degradative capabilities and the ability to thrive in the presence of surfactants. The guts of some marine invertebrates, particularly deposit feeders, have previously been shown to contain high levels of biosurfactants. Our primary aim was to mine these natural, surfactant-rich habitats for surfactant-resistant bacteria. Relative to sediment porewaters, the gut contents of two polychaete deposit feeders,Nereis succineaandAmphitrite ornata, exhibited a significantly higher ratio of bacteria resistant to both cationic and anionic surfactants. In contrast, bacteria in the gut fluids of a holothuroid,Leptosynapta tenuis, showed surfactant susceptibility similar to that of bacteria from sediments. Analyses of 16S rRNA gene sequences revealed that the majority of surfactant-resistant isolates were previously undescribed species of the genusVibrioor were of a group most closely related toSpongiobacterspp. We also tested a subset of resistant bacteria for the production of biosurfactants. The majority did produce biosurfactants, as demonstrated via the oil-spreading method, but in all cases, production was relatively weak under the culture conditions employed. Novel surfactant-resistant, biosurfactant-producing bacteria, and the habitats from which they were isolated, provide a new source pool for potential microorganisms to be exploited in the in situ bioremediation of marine sediments.
Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in
Tetrahymena thermophila
Authors: Wloga Dorota , Rogowski Krzysztof , Sharma Neeraj , Van Dijk Juliette , Janke Carsten , Eddé Bernard , Bré Marie-Hélène , Levilliers Nicolette , Redeker Virginie , Duan Jianming , Gorovsky Martin A. , Jerka-Dziadosz Maria , Gaertig Jacek 
Published: June 2008

ABSTRACT

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate
Tetrahymena
, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the α-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of α-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as α-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the
TTLL1
and
TTLL9
genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on α-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.
Microtubule Motor Protein Kar3 Is Required for Normal Mitotic Division and Morphogenesis in
Candida albicans
Authors: Sherwood Racquel Kim , Bennett Richard J. 
Published: June 2008

ABSTRACT

The kinesin-related protein Kar3 is a minus end-directed molecular motor that plays a multifunctional role in microtubule-directed nuclear movement. Previously, it was shown that
Candida albicans
Kar3p is critical for nuclear fusion during mating as
kar3
mutants were defective in karyogamy. In this study, we confirm that Kar3p is required for nuclear congression in mating but that neither Kar3p nor the dynein motor protein Dyn1p is required for nuclear migration in the mating projection prior to cell fusion. In addition, we show that
C
.
albicans
Kar3p plays an important role in the cell and colony morphology of mitotically dividing cells, as evidenced by diminished filamentation of
kar3
cells on Spider medium and an increased tendency of mutant cells to form pseudohyphal cells in liquid culture. Loss of Kar3p also led to defects in nuclear division, causing cells to grow slowly and exhibit reduced viability compared to wild-type cells. Slow growth was due, at least in part, to delayed cell cycle progression, as cells lacking Kar3p accumulated in anaphase of the cell cycle. Consistent with a role in mitotic division, Kar3 protein was shown to localize to the spindle pole bodies. Finally,
kar3
cells exhibited unstable or aberrant mitotic spindles, a finding that accounts for the delay in cell cycle progression and decreased viability of these cells. We suggest that the altered morphology of
kar3
cells is a direct consequence of the delay in anaphase, and this leads to increased polarized growth and pseudohypha formation.
Neurospora crassa heat shock factor 1
Is an Essential Gene; a Second Heat Shock Factor-Like Gene,
hsf2
, Is Required for Asexual Spore Formation
Authors: Thompson Seona , Croft Nirvana J. , Sotiriou Antonis , Piggins Hugh D. , Crosthwaite Susan K. 
Published: June 2008

ABSTRACT

Appropriate responses of organisms to heat stress are essential for their survival. In eukaryotes, adaptation to high temperatures is mediated by heat shock transcription factors (HSFs). HSFs regulate the expression of heat shock proteins, which function as molecular chaperones assisting in protein folding and stability. In many model organisms a great deal is known about the products of
hsf
genes. An important exception is the filamentous fungus and model eukaryote
Neurospora crassa
. Here we show that two
Neurospora crassa
genes whose protein products share similarity to known HSFs play different biological roles. We report that
heat shock factor 1
(
hsf1
) is an essential gene and that
hsf2
is required for asexual development. Conidiation may be blocked in the
hsf2
knockout (
hsf2

KO

) strain because HSF2 is an integral element of the conidiation pathway or because it affects the availability of protein chaperones. We report that genes expressed during conidiation, for example
fluffy
,
conidiation-10
, and
repressor of conidiation-1
show wild-type levels of expression in a
hsf2

KO

strain. However, consistent with the lack of macroconidium development, levels of
eas
are much reduced. Cultures of the
hsf2

KO

strain along with two other aconidial strains, the
fluffy
and
aconidial-2
strains, took longer than the wild type to recover from heat shock. Altered expression profiles of
hsp90
and a putative
hsp90
-associated protein in the
hsf2

KO

strain after exposure to heat shock may in part account for its reduced ability to cope with heat stress.
Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm
Authors: Savic Miloje , Ilic-Tomic Tatjana , Macmaster Rachel , Vasiljevic Branka , Conn Graeme L. 
Published: June 2008

ABSTRACTThe 16S rRNA methyltransferase Sgm from “Micromonospora zionensis” confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of theS-adenosyl-l-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with aKD(binding constant) of 17.6 μM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.
Loss of Flagellum-Based Motility by
Listeria monocytogenes
Results in Formation of Hyperbiofilms
Authors: Todhanakasem Tatsaporn , Young Glenn M. 
Published: June 2008

ABSTRACT

Biofilm formation by the gram-positive, motile, food-borne pathogen
Listeria monocytogenes
was demonstrated to occur by an ordered series of stages. Biofilm development involves flagellum-based motility, which when blocked decreases initial bacterial surface attachment but subsequently leads to the formation of hyperbiofilms, surface-attached communities reaching high density.
Genomic O Island 122, Locus for Enterocyte Effacement, and the Evolution of Virulent Verocytotoxin-ProducingEscherichia coli
Authors: Konczy Paulina , Ziebell Kim , Mascarenhas Mariola , Choi Aileen , Michaud Corinne , Kropinski Andrew M. , Whittam Thomas S. , Wickham Mark , Finlay Brett , Karmali Mohamed A. 
Published: June 2008

ABSTRACTThe locus of enterocyte effacement (LEE) and genomic O island 122 (OI-122) are pathogenicity islands in verocytotoxin-producingEscherichia coli(VTEC) serotypes that are associated with outbreaks and serious disease. Composed of three modules, OI-122 may occur as “complete” (with all three modules) or “incomplete” (with one or two modules) in different strains. OI-122 encodes two non-LEE effector (Nle) molecules that are secreted by the LEE type III secretion system, and LEE and OI-122 are cointegrated in some VTEC strains. Thus, they are functionally linked, but little is known about the patterns of acquisition of these codependent islands. To examine this, we conducted a population genetics analysis, using multilocus sequence typing (MLST), with 72 VTEC strains (classified into seropathotypes A to E) and superimposed on the results the LEE and OI-122 contents of these organisms. The wide distribution of LEE and OI-122 modules among MLST clonal groups corroborates the hypothesis that there has been lateral transfer of both pathogenicity islands. Sequence analysis of apagC-like gene in OI-122 module 1 also revealed two nonsynonymous single-nucleotide polymorphisms that could help discriminate a subset of seropathotype C strains and determine the presence of the LEE. A nonsense mutation was found in this gene in five less virulent strains, consistent with a decaying or inactive gene. The modular nature of OI-122 could be explained by the acquisition of modules by lateral transfer, either singly or as a group, and by degeneration of genes within modules. Correlations between clonal group, seropathotype, and LEE and OI-122 content provide insight into the role of genomic islands in VTEC evolution.
The Striking Case of Tryptophan Provision in the Cedar Aphid
Cinara cedri
Authors: Gosalbes María José , Lamelas Araceli , Moya Andrés , Latorre Amparo 
Published: June 2008

ABSTRACT

Buchnera aphidicola
BCc has lost its symbiotic role as the tryptophan supplier to the aphid
Cinara cedri
. We report the presence of a plasmid in this endosymbiont that contains the
trpEG
genes. The remaining genes for the pathway (
trpDCBA
) are located on the chromosome of the secondary endosymbiont “
Candidatus
Serratia symbiotica.” Thus, we propose that a symbiotic consortium is necessary to provide tryptophan.
Cloning and Characterization of the Tetrocarcin A Gene Cluster from
Micromonospora chalcea
NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics
Authors: Fang Jie , Zhang Yiping , Huang Lijuan , Jia Xinying , Zhang Qi , Zhang Xu , Tang Gongli , Liu Wen 
Published: June 2008

ABSTRACT

Tetrocarcin A (TCA), produced by
Micromonospora chalcea
NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of
d
-tetronitrose,
l
-amicetose, and
l
-digitoxose may begin with
d
-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and
trans
-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene
tcaD3
(with the functions of
chlD3
and
chlD4
) to Δ
chlD3
and Δ
chlD4
in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.
CcpN Controls Central Carbon Fluxes in
Bacillus subtilis
Authors: Tännler Simon , Fischer Eliane , Le Coq Dominique , Doan Thierry , Jamet Emmanuel , Sauer Uwe , Aymerich Stéphane 
Published: June 2008

ABSTRACT

The transcriptional regulator CcpN of
Bacillus subtilis
has been recently characterized as a repressor of two gluconeogenic genes,
gapB
and
pckA
, and of a small noncoding regulatory RNA,
sr1
, involved in arginine catabolism. Deletion of
ccpN
impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose,
gapB
and
pckA
are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of
pckA
under glycolytic condition causes the growth defect observed in the
ccpN
mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a
ccpN
mutant. The high flux through the PP pathway in the
ccpN
mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of
sr1
, the third known target of CcpN, plays only a marginal role in
ccpN
mutant phenotypes.
Vibrio choleraeVciB Promotes Iron Uptake via Ferrous Iron Transporters
Authors: Mey Alexandra R. , Wyckoff Elizabeth E. , Hoover Lindsey A. , Fisher Carolyn R. , Payne Shelley M. 
Published: June 2008

ABSTRACTVibrio choleraeuses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein inV. cholerae, VciB. ThevciBgene and its linked gene,vciA, were isolated in a screen forV. choleraegenes that permitted growth of anEscherichia colisiderophore mutant in low-iron medium. ThevciABoperon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation ofE. coliandShigella flexneristrains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen whenvciBwas expressed in anE. coliorS. flexneristrain defective for the ferrous iron transporter Feo. Supplying theE. coli feomutant with a plasmid encoding eitherE. coliorV. choleraeFeo, or theS. flexneriferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systemsV. choleraeFbp andHaemophilus influenzaeHit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. AV. cholerae vciBmutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.
Conversion of
Bacillus subtilis
OhrR from a 1-Cys to a 2-Cys Peroxide Sensor
Authors: Soonsanga Sumarin , Lee Jin-Won , Helmann John D. 
Published: June 2008

ABSTRACT

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by
Bacillus subtilis
OhrR) and 2-Cys (represented by
Xanthomonas campestris
OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The
B. subtilis
1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the
X. campestris
2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys
B. subtilis
OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the
X. campestris
OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.
Role of Spore Coat Proteins in the Resistance of
Bacillus subtilis
Spores to
Caenorhabditis elegans
Predation
Authors: Laaberki Maria-Halima , Dworkin Jonathan 
Published: June 2008

ABSTRACT

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode
Caenorhabditis elegans
, a likely predator of
Bacillus
spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type
Bacillus subtilis
spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using
B. subtilis
strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to
C. elegans
digestion depending on the extent of the spore coat structure modifications.
The Plant-Associated BacteriumStenotrophomonas rhizophilaExpresses a New Enzyme for the Synthesis of the Compatible Solute Glucosylglycerol
Authors: Hagemann Martin , Ribbeck-Busch Kathrin , Klähn Stephan , Hasse Dirk , Steinbruch Robert , Berg Gabriele 
Published: June 2008

ABSTRACTThe rhizobacteriumStenotrophomonas rhizophilaaccumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme fromS. rhizophilarepresented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned fromPseudomonassp. strain OA146. TheggpPSgene was induced after a salt shock inS. rhizophilacells. After the salt-loaded cells reached stationary phase, theggpPSmRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The completeggpPSgene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacteriumSynechocystissp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis inS. rhizophilawere found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.