bluuebunny/crossref_metadata_2025 huggingface dataset:
Epsilonproteobacteria
Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas
Authors: Grote Jana , Jost Günter , Labrenz Matthias , Herndl Gerhard J. , Jürgens Klaus
Published: October 2008

Recent studies have indicated that chemoautotrophic
Epsilonproteobacteria
might play an important role, especially as anaerobic or microaerophilic dark CO
2
-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO
2
-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of
Epsilonproteobacteria
to dark CO
2
fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [
14
C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10
5
14
CO
2
-fixing prokaryotic cells ml
−1
were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10
4
14
CO
2
-fixing cells ml
−1
were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance.
14
CO
2
-incorporating cells belonged exclusively to the domain
Bacteria
. Among these, members of the
Epsilonproteobacteria
were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the
Sulfurimonas
subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO
2
-fixing group. In conclusion,
Epsilonproteobacteria
were found to be mainly responsible for chemoautotrophic activity in the dark CO
2
fixation maxima of the Black Sea and central Baltic Sea redoxclines. These
Epsilonproteobacteria
might be relevant in similar habitats of the world's oceans, where high dark CO
2
fixation rates have been measured.
Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude
Escherichia coli
Cell Extracts in Response to Copper Ions
Authors: Thieme Daniel , Neubauer Peter , Nies Dietrich H. , Grass Gregor
Published: October 2008

Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from
Escherichia coli
without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of
E. coli
. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.
Rapid Classification and Identification of Salmonellae at the Species and Subspecies Levels by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Authors: Dieckmann Ralf , Helmuth Reiner , Erhard Marcel , Malorny Burkhard
Published: October 2008

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing
Salmonella enterica
subsp.
enterica
(subspecies I),
S. enterica
subsp.
salamae
(subspecies II),
S. enterica
subsp.
arizonae
(subspecies IIIa),
S. enterica
subsp.
diarizonae
(subspecies IIIb),
S. enterica
subsp.
houtenae
(subspecies IV), and
S. enterica
subsp.
indica
(subspecies VI), and
Salmonella bongori
were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for
Salmonella
, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of
Salmonella
strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of
Salmonella
subspecies in minutes was established.
Control of Lipid Accumulation in the Yeast
Yarrowia lipolytica
Authors: Beopoulos Athanasios , Mrozova Zuzana , Thevenieau France , Le Dall Marie-Thérèse , Hapala Ivan , Papanikolaou Seraphim , Chardot Thierry , Nicaud Jean-Marc
Published: October 2008

A genomic comparison of
Yarrowia lipolytica
and
Saccharomyces cerevisiae
indicates that the metabolism of
Y. lipolytica
is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the
GUT2
gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in
Y. lipolytica
in this study. This Δ
gut2
mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to β-oxidation.
Y. lipolytica
contains six acyl-coenzyme A oxidases (Aox), encoded by the
POX1
to
POX6
genes, that catalyze the limiting step of peroxisomal β-oxidation. Additional deletion of the
POX1
to
POX6
genes in the Δ
gut2
strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.
Possible Involvement of an Extracellular Superoxide Dismutase (SodA) as a Radical Scavenger in Poly(
cis
-1,4-Isoprene) Degradation
Authors: Schulte Carina , Arenskötter Matthias , Berekaa Mahmoud M. , Arenskötter Quyen , Priefert Horst , Steinbüchel Alexander
Published: October 2008

Gordonia westfalica
Kb1 and
Gordonia polyisoprenivorans
VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(
cis
-1,4-isoprene) degradation. To investigate the function of this enzyme in
G. polyisoprenivorans
VH2, the
sodA
gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(
cis
-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiments revealed that SodA activity is required for optimal growth on poly(
cis
-1,4-isoprene), whereas this enzyme has no effect on aerobic growth in the presence of water-soluble substrates like succinate, acetate, and propionate. This was detected by activity staining, and proof of expression was by antibody detection of SOD. When SodA from
G. westfalica
Kb1 was heterologously expressed in the
sodA sodB
double mutant
Escherichia coli
QC779, the recombinant mutant exhibited increased resistance to paraquat, thereby indicating the functionality of the
G. westfalica
Kb1 SodA and indirectly protection of
G. westfalica
cells by SodA from oxidative damage. Both
sodA
from
G. polyisoprenivorans
VH2 and
sodA
from
G. westfalica
Kb1 coded for polypeptides comprising 209 amino acids and having approximately 90% and 70% identical amino acids, respectively, to the SodA from
Mycobacterium smegmatis
strain MC
2
155 and
Micrococcus luteus
NCTC 2665. As revealed by activity staining experiments with the wild type and the disruption mutant of
G. polyisoprenivorans
, this bacterium harbors only one active SOD belonging to the manganese family. The N-terminal sequences of the extracellular SodA proteins of both
Gordonia
species showed no evidence of leader peptides for the mature proteins, like the intracellular SodA protein of
G. polyisoprenivorans
VH2, which was purified under native conditions from the cells. In
G. westfalica
Kb1 and
G. polyisoprenivorans
VH2, SodA probably provides protection against reactive oxygen intermediates which occur during degradation of poly(
cis
-1,4-isoprene).
Isolation of New
Stenotrophomonas
Bacteriophages and Genomic Characterization of Temperate Phage S1
Authors: García Pilar , Monjardín Cristina , Martín Rebeca , Madera Carmen , Soberón Nora , Garcia Eva , Meana Álvaro , Suárez Juan E.
Published: October 2008

Twenty-two phages that infect
Stenotrophomonas
species were isolated through sewage enrichment and prophage induction. Of them, S1, S3, and S4 were selected due to their wide host ranges compared to those of the other phages. S1 and S4 are temperate siphoviruses, while S3 is a virulent myovirus. The genomes of S3 and S4, about 33 and 200 kb, were resistant to restriction digestion. The lytic cycles lasted 30 min for S3 and about 75 min for S1 and S4. The burst size for S3 was 100 virions/cell, while S1 and S4 produced about 75 virus particles/cell. The frequency of bacteriophage-insensitive host mutants, calculated by dividing the number of surviving colonies by the bacterial titer of a parallel, uninfected culture, ranged between 10
−5
and 10
−6
for S3 and 10
−3
and 10
−4
for S1 and S4. The 40,287-bp genome of S1 contains 48 open reading frames (ORFs) and 12-bp 5′ protruding cohesive ends. By using a combination of bioinformatics and experimental evidence, functions were ascribed to 21 ORFs. The morphogenetic and lysis modules are well-conserved, but no lysis-lysogeny switch or DNA replication gene clusters were recognized. Two major clusters of genes with respect to transcriptional orientation were observed. Interspersed among them were lysogenic conversion genes encoding phosphoadenosine phosphosulfate reductase and GspM, a protein involved in the general secretion system II. The
attP
site of S1 may be located within a gene that presents over 75% homology to a
Stenotrophomonas
chromosomal determinant.
Phaeobacter
and
Ruegeria
Species of the
Roseobacter
Clade Colonize Separate Niches in a Danish Turbot (
Scophthalmus maximus
)-Rearing Farm and Antagonize
Vibrio anguillarum
under Different Growth Conditions
Authors: Porsby Cisse Hedegaard , Nielsen Kristian Fog , Gram Lone
Published: October 2008

Members of the
Roseobacter
clade colonize a Spanish turbot larval unit, and one isolate (
Phaeobacter
strain 27-4) is capable of disease suppression in in vivo challenge trials. Here, we demonstrate that roseobacters with antagonistic activity against
Vibrio anguillarum
also colonize a Danish turbot larval farm that relies on a very different water source (the Danish fiord Limfjorden as opposed to the Galician Atlantic Ocean). Phylogenetic analyses based on 16S rRNA and gyrase B gene sequences revealed that different species colonized different niches in the larval unit.
Phaeobacter inhibens-
and
Phaeobacter gallaeciensis
-like strains were primarily found in the production sites, whereas strains identified as
Ruegeria mobilis
or
Ruegeria pelagia
were found only in the algal cultures.
Phaeobacter
spp. were more inhibitory against the general microbiota from the Danish turbot larval unit than were the
Ruegeria
spp.
Phaeobacter
spp. produced tropodithietic acid (TDA) and brown pigment and antagonized
V. anguillarum
when grown under shaking (200 rpm) and stagnant (0 rpm) conditions, whereas
Ruegeria
spp. behaved similarly to
Phaeobacter
strain 27-4 and expressed these three phenotypes only during stagnant growth. Both genera attached to an inert surface and grew in multicellular rosettes after stagnant growth, whereas shaking conditions led to single cells with low attachment capacity. Bacteria from the
Roseobacter
clade appear to be universal colonizers of marine larval rearing units, and since the Danish
Phaeobacter
spp. displayed antibacterial activity under a broader range of growth conditions than did
Phaeobacter
strain 27-4, these organisms may hold greater promise as fish probiotic organisms.
Loss of Gibberellin Production in
Fusarium verticillioides
(
Gibberella fujikuroi
MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster
Authors: Bömke Christiane , Rojas Maria C. , Hedden Peter , Tudzynski Bettina
Published: October 2008

Fusarium verticillioides
(
Gibberella fujikuroi
mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the
Gibberella fujikuroi
species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to
Fusarium fujikuroi
(
G. fujikuroi
MP-C) and
Fusarium konzum
(MP-I), although most members of the
G. fujikuroi
species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in
F. verticillioides
(MP-A) is due to the loss of a majority of the GA gene cluster as found in
F. fujikuroi
. The remaining part of the cluster consists of the full-length
F. verticillioides des
gene (Fv
des
), encoding the GA
4
desaturase, and the coding region of Fv
P450-4
, encoding the
ent
-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of
F. fujikuroi
. The GA production capacity of
F. verticillioides
was restored by transforming a cosmid with the entire GA gene cluster from
F. fujikuroi
, indicating the existence of an active regulation system in
F. verticillioides
. Furthermore, the GA
4
desaturase gene
des
from
F. verticillioides
encodes an active enzyme which was able to restore the GA production in a corresponding
des
deletion mutant of
F. fujikuroi
.
Enhanced Transformation Efficiency of Recalcitrant
Bacillus cereus
and
Bacillus weihenstephanensis
Isolates upon In Vitro Methylation of Plasmid DNA
Authors: Groot Masja Nierop , Nieboer Frank , Abee Tjakko
Published: October 2008

Digestion patterns of chromosomal DNAs of
Bacillus cereus
and
Bacillus weihenstephanensis
strains suggest that Sau3AI-type restriction modification systems are widely present among the isolates tested. In vitro methylation of plasmid DNA was used to enhance poor plasmid transfer upon electroporation to recalcitrant strains that carry Sau3AI restriction barriers.
Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on
Escherichia coli
Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity
Authors: Yang Chao , Zhu Yaran , Yang Jijian , Liu Zheng , Qiao Chuanling , Mulchandani Ashok , Chen Wilfred
Published: October 2008

Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of
Escherichia coli
by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered
E. coli
can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered
E. coli
and a natural
p
-nitrophenol (PNP) degrader,
Ochrobactrum
sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.
In Vivo
Himar1
Transposon Mutagenesis of
Burkholderia pseudomallei
Authors: Rholl Drew A. , Trunck Lily A. , Schweizer Herbert P.
Published: October 2008

Burkholderia psedudomallei
is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a
Himar1
-based random mutagenesis system for
B. pseudomallei
(
HimarBP
). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent
B. pseudomallei
strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal
HimarBP
insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued
HimarBP
insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways.
HimarBP
insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with
B. thailandensis
suggest that the newly developed
HimarBP
transposons can also be used for random mutagenesis of other
Burkholderia
spp., especially the closely related species
B. mallei
. Our results demonstrate that comprehensive transposon libraries of
B. pseudomallei
can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.
Directed Evolution of
Methanococcus jannaschii
Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by
Escherichia coli
Authors: Atsumi Shota , Liao James C.
Published: October 2008

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from
Escherichia coli
using a 2-keto acid-based pathway. Here, we took advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from
Methanococcus jannaschii
. This new pathway, which directly converts pyruvate to 2-ketobutyrate, bypasses threonine biosynthesis and represents the shortest keto acid-mediated pathway for producing 1-propanol and 1-butanol from glucose. Directed evolution of CimA enhanced the specific activity over a wide temperature range (30 to 70°C). The best CimA variant was found to be insensitive to feedback inhibition by isoleucine in addition to the improved activity. This CimA variant enabled 9- and 22-fold higher production levels of 1-propanol and 1-butanol, respectively, compared to the strain expressing the wild-type CimA. This work demonstrates (i) the first production of 1-propanol and 1-butanol using the citramalate pathway and (ii) the benefit of the 2-keto acid pathway that enables a growth-based evolutionary strategy to improve the production of non-growth-related products.
Stress-Induced Cell Death Is Mediated by Ceramide Synthesis in
Neurospora crassa
Authors: Plesofsky Nora S. , Levery Steven B. , Castle Sherry A. , Brambl Robert
Published: October 2008

The combined stresses of moderate heat shock (45°C) and analog-induced glucose deprivation constitute a lethal stress for
Neurospora crassa
. We found that this cell death requires fatty acid synthesis and the cofactor biotin. In the absence of the cofactor, the stressed cells are particularly sensitive to exogenous ceramide, which is lethal at low concentrations. When we extracted endogenous sphingolipids, we found that unique ceramides were induced (i) by the inhibitory glucose analog 2-deoxyglucose and (ii) by combined heat shock and 2-deoxyglucose. We determined that the former is a 2-deoxyglucose-modified ceramide. By structural analysis, we identified the latter, induced by dual stress, as C
18
(OH)-phytoceramide. We also identified C
24
(OH)-phytoceramide as a constitutive ceramide that continues to be produced during the combined stresses. The unusual C
18
(OH)-phytoceramide is not made by germinating asexual spores subjected to the same heat and carbon stress. Since these spores, unlike growing cells, do not die from the stresses, this suggests a possible connection between synthesis of the dual-stress-induced ceramide and cell death. This connection is supported by the finding that a (dihydro)ceramide synthase inhibitor, australifungin, renders cells resistant to death from these stresses. The OS-2 mitogen-activated protein kinase, homologous to mammalian p38, may be involved in the cell death signaling pathway. Strains lacking OS-2 survived the combined stresses better than the wild type, and phosphorylated OS-2 increased in wild-type cells in response to heat shock and combined heat and carbon stress.
Localization and Interaction of the Proteins Constituting the
GAL
Genetic Switch in
Saccharomyces cerevisiae
Authors: Wightman Raymond , Bell Rachel , Reece Richard J.
Published: October 2008

In
Saccharomyces cerevisiae
, the
GAL
genes encode the enzymes required for galactose metabolism. Regulation of these genes has served as the paradigm for eukaryotic transcriptional control over the last 50 years. The switch between inert and active gene expression is dependent upon three proteins—the transcriptional activator Gal4p, the inhibitor Gal80p, and the ligand sensor Gal3p. Here, we present a detailed spatial analysis of the three
GAL
regulatory proteins produced from their native genomic loci. Using a novel application of photobleaching, we demonstrate, for the first time, that the Gal3p ligand sensor enters the nucleus of yeast cells in the presence of galactose. Additionally, using Förster resonance energy transfer, we show that the interaction between Gal3p and Gal80p occurs throughout the yeast cell. Taken together, these data challenge existing models for the cellular localization of the regulatory proteins during the induction of
GAL
gene expression by galactose and suggest a mechanism for the induction of the
GAL
genes in which galactose-bound Gal3p moves from the cytoplasm to the nucleus to interact with the transcriptional inhibitor Gal80p.
Insight into the Role of HOG Pathway Components Ssk2p, Pbs2p, and Hog1p in the Opportunistic Yeast
Candida lusitaniae
Authors: Boisnard Stéphanie , Ruprich-Robert Gwenaël , Florent Martine , Da Silva Bruno , Chapeland-Leclerc Florence , Papon Nicolas
Published: October 2008

In the present study, we have investigated the role of
SSK2
,
PBS2
, and
HOG1
, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in
Candida lusitaniae
. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.
Capsular Localization of theCryptococcus neoformansPolysaccharide Component Galactoxylomannan
Authors: De Jesus Magdia , Nicola André Moraes , Rodrigues Marcio L. , Janbon Guilhem , Casadevall Arturo
Published: October 2008

Cryptococcus neoformanscapsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in theC. neoformanscapsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen fromBacillus anthracisas a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.
Cytoplasmic Protein Mobility in Osmotically Stressed
Escherichia coli
Authors: Konopka Michael C. , Sochacki Kem A. , Bratton Benjamin P. , Shkel Irina A. , Record M. Thomas , Weisshaar James C.
Published: October 2008

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably,
Escherichia coli
grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction (〈φ〉) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar 〈φ〉 range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (
D
GFP
) in the cytoplasm of
E. coli
cells as a function of 〈φ〉 for both plasmolyzed and adapted cells. For plasmolyzed cells, the median
D
GFP
(

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) decreases by a factor of 70 as 〈φ〉 increases from 0.16 to 0.33. In sharp contrast, for adapted cells,

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decreases only by a factor of 2.1 as 〈φ〉 increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by 〈φ〉 alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.
Conserved Residues in the HAMP Domain Define a New Family of Proposed Bipartite Energy Taxis Receptors
Authors: Elliott Kathryn T. , Zhulin Igor B. , Stuckey Jeanne A. , DiRita Victor J.
Published: October 2008

HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to those of the Aer energy taxis receptor of
Escherichia coli
, which has both a HAMP domain and a sensory PAS domain.
Campylobacter jejuni
has an energy taxis system consisting of the domains of Aer divided between two proteins, CetA (HAMP domain containing) and CetB (PAS domain containing). In this study, we found that the CetA HAMP domain differs significantly from that of Aer in the predicted secondary structure. Using similarity searches, we identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Within these proteins, we identified nine residues in the HAMP domain and proximal signaling domain that are highly conserved, at least three of which are required for CetA function. Additionally, we demonstrated that CetA contributes to the invasion of human epithelial cells by
C. jejuni
, while CetB does not. This finding supports the hypothesis that members of HAMP/PAS pairs possess the capacity to act independently of each other in cellular traits other than energy taxis.
An Unexpected Effect of Tetracycline Concentration: Growth Phase-Associated Excision of theBacteroidesMobilizable Transposon NBU1
Authors: Song Bo , Wang Gui-Rong , Shoemaker Nadja B. , Salyers Abigail A.
Published: October 2008

Early studies of theBacteroidesmobilizable transposon NBU1 established that excision, the first step in NBU1 transfer, requires exposure of the cells to tetracycline. More recently, we found that excision is also associated with growth phase; even after exposure to tetracycline, excision is detectable only after the cells enter late exponential phase. The tetracycline effect is mediated by a two-component regulatory system, RteA and RteB, which is provided intransby an integrated self-transmissible element, CTnDOT. TherteAandrteBgenes are part of a three-gene operon that also contains the tetracycline resistance genetetQ. We report here that neither transcription nor translation of thetetQ-rteA-rteBoperon is affected by growth phase. Moreover, RteA is not required for the growth phase effect, because a mutant form of RteB that does not require phosphorylation by RteA did not make excision independent of growth phase. Two conditions made NBU1 excision independent of growth phase. One was reducing the tetracycline concentration from an inhibitory concentration (1 μg/ml) to a subinhibitory level (0.05 μg/ml). Independence of growth phase also occurred whenrteAandrteBwere placed under the control of a heterologous maltose-inducible promoter, PsusA. Our results suggest that at low concentrations of tetracycline, ribosomes are capable of translating enough RteA and RteB for excision to occur. At higher tetracycline concentrations, however, TetQ is needed to protect enough ribosomes to allow the translation of excision genes, and this protection takes time to develop. Thus, subinhibitory concentrations of tetracycline may increase the probability of gene transfer because, in contrast to inhibitory concentrations, excision can occur at all phases of growth.
Interaction between Bacteriophage DMS3 and Host CRISPR Region Inhibits Group Behaviors ofPseudomonas aeruginosa
Authors: Zegans Michael E. , Wagner Jeffrey C. , Cady Kyle C. , Murphy Daniel M. , Hammond John H. , O'Toole George A.
Published: October 2008

Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) andcas(CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection ofPseudomonas aeruginosaPA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the sixcasgenes and one of the two CRISPRs in this region restored biofilm formation and swarming to DMS3 lysogenized strains. Our observations suggest a role for CRISPR regions in modifying the effects of lysogeny onP. aeruginosa.
The AsaP1 Peptidase of
Aeromonas salmonicida
subsp.
achromogenes
Is a Highly Conserved Deuterolysin Metalloprotease (Family M35) and a Major Virulence Factor
Authors: Arnadottir Helga , Hvanndal Iris , Andresdottir Valgerdur , Burr Sarah E. , Frey Joachim , Gudmundsdottir Bjarnheidur K.
Published: October 2008

Infections by the bacterium
Aeromonas salmonicida
subsp.
achromogenes
cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by
A. salmonicida
subsp.
achromogenes
, belongs to the group of extracellular peptidases (
Aeromonas
type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Δ
asaP1 A. salmonicida
subsp.
achromogenes
strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Δ
asaP1
strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.
The Small RNA GcvB Regulates
sstT
mRNA Expression in
Escherichia coli
Authors: Pulvermacher Sarah C. , Stauffer Lorraine T. , Stauffer George V.
Published: October 2008

In
Escherichia coli
, the
gcvB
gene encodes a nontranslated RNA (referred to as GcvB) that regulates OppA and DppA, two periplasmic binding proteins for the oligopeptide and dipeptide transport systems. An additional regulatory target of GcvB,
sstT
, was found by microarray analysis of RNA isolated from a wild-type strain and a
gcvB
deletion strain grown to mid-log phase in Luria-Bertani broth. The SstT protein functions to transport
l
-serine and
l
-threonine by sodium transport into the cell. Reverse transcription-PCR and translational fusions confirmed that GcvB negatively regulates
sstT
mRNA levels in cells grown in Luria-Bertani broth. A series of transcriptional fusions identified a region of
sstT
mRNA upstream of the ribosome binding site needed for negative regulation by GcvB. Analysis of the GcvB RNA identified a sequence complementary to this region of the
sstT
mRNA. The region of GcvB complementary to
sstT
mRNA is the same region of GcvB identified to regulate the
dppA
and
oppA
mRNAs. Mutations predicted to disrupt base pairing between
sstT
mRNA and GcvB were made in
gcvB
, which resulted in the identification of a small region of GcvB necessary for negative regulation of
sstT-lacZ
. Additionally, the RNA chaperone protein Hfq was found to be necessary for GcvB to negatively regulate
sstT
-
lacZ
in Luria-Bertani broth and glucose minimal medium supplemented with glycine. The
sstT
mRNA is the first target found to be regulated by GcvB in glucose minimal medium supplemented with glycine.
The
Agrobacterium rhizogenes
GALLS Gene Encodes Two Secreted Proteins Required for Genetic Transformation of Plants
Authors: Hodges Larry D. , Lee Lan-Ying , McNett Henry , Gelvin Stanton B. , Ream Walt
Published: October 2008

Agrobacterium tumefaciens
and
Agrobacterium rhizogenes
are related pathogens that cause crown gall and hairy root diseases, which result from integration and expression of bacterial genes in the plant genome. Single-stranded DNA (T strands) and virulence proteins are translocated into plant cells by a type IV secretion system. VirD2 nicks a specific DNA sequence, attaches to the 5′ end, and pilots the DNA into plant cells.
A. tumefaciens
translocates single-stranded DNA-binding protein VirE2 into plant cells where it likely binds T strands and may aid in targeting them into the nucleus. Although some
A. rhizogenes
strains lack VirE2, they transfer T strands efficiently due to the GALLS gene, which complements an
A. tumefaciens virE2
mutant for tumor formation. Unlike VirE2, full-length GALLS (GALLS-FL) contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. GALLS-FL and VirE2 contain nuclear localization signals (NLS) and secretion signals. Mutations in any of these domains abolish the ability of the GALLS gene to substitute for v
irE2
. Here, we show that the GALLS gene encodes two proteins from one open reading frame: GALLS-FL and a protein comprised of the C-terminal domain, which initiates at an internal in-frame start codon. On some hosts, both GALLS proteins were required to substitute for VirE2. GALLS-FL tagged with yellow fluorescent protein localized to the nucleus of tobacco cells in an NLS-dependent manner. In plant cells, the GALLS proteins interacted with themselves, VirD2, and each other. VirD2 interacted with GALLS-FL and localized inside the nucleus, where its predicted helicase activity may pull T strands into the nucleus.
Four VirB6 Paralogs and VirB9 Are Expressed and Interact inEhrlichia chaffeensis-Containing Vacuoles
Authors: Bao Weichao , Kumagai Yumi , Niu Hua , Yamaguchi Mamoru , Miura Koshiro , Rikihisa Yasuko
Published: October 2008

The type IV secretion system is an important virulence factor in several host cell-associated pathogens, as it delivers various bacterial macromolecules to target eukaryotic cells. Genes homologous to severalvirBgenes andvirD4ofAgrobacterium tumefaciensare found in an intravacuolar pathogenEhrlichia chaffeensis, the tick-borne causative agent of human monocytic ehrlichiosis. In particular, despite its small genome size,E. chaffeensishas four tandemvirB6paralogs (virB6-1, -2, -3, and -4) that are 3- to 10-fold larger thanA. tumefaciens virB6. The present study for the first time illustrates the relevance of the larger quadruple VirB6 paralogs by demonstrating the protein expression and interaction inE. chaffeensis. All fourvirB6paralogs were cotranscribed in THP-1 human leukemia and ISE6 tick cell cultures. The four VirB6 proteins and VirB9 were expressed byE. chaffeensisin THP-1 cells, and amounts of these five proteins were similar in isolatedE. chaffeensis-containing vacuoles and vacuole-freeE. chaffeensis. In addition, an 80-kDa fragment of VirB6-2 was detected, which was strikingly more prevalent inE. chaffeensis-containing vacuoles than in vacuole-freeE. chaffeensis. Coimmunoprecipitation analysis revealed VirB9 interaction with VirB6-1 and VirB6-2; VirB6-4 interaction with VirB6-1, VirB6-2, and VirB6-3; and VirB6-2 80-kDa fragment interaction with VirB6-3 and VirB6-4. The interaction of VirB9 and VirB6-2 was confirmed by far-Western blotting. The results suggest thatE. chaffeensisVirB9, the quadruple VirB6 proteins, and the VirB6-2 80-kDa fragment form a unique molecular subassembly to cooperate in type IV secretion.
The GAF-Like-Domain-Containing Transcriptional Regulator DfdR Is a Sensor Protein for Dibenzofuran and Several Hydrophobic Aromatic Compounds
Authors: Iida Toshiya , Waki Taro , Nakamura Kaoru , Mukouzaka Yuki , Kudo Toshiaki
Published: October 2008

Dibenzofuran (DF) is one of the dioxin carbon skeletal compounds used as a model to study the microbial degradation of dioxins. This study analyzed the transcriptional regulation of the DF dioxygenase genes
dfdA1
to
dfdA4
in the DF-utilizing actinomycetes
Rhodococcus
sp. strain YK2 and
Terrabacter
sp. strain YK3. An open reading frame designated
dfdR
was detected downstream of the
dfdC
genes. The C-terminal part of the DfdR amino acid sequence has high levels of similarity to several LuxR-type DNA binding helix-turn-helix domains, and a GAF domain sequence in the central part was detected by a domain search analysis. A derivative of YK2 with
dfdR
disrupted was not able to utilize DF and did not exhibit DF-dependent
dfdA1
transcriptional induction ability, and these dysfunctions were compensated for by introduction of
dfdR
. Promoter analysis of
dfdA1
in
Rhodococcus
strains indicated that activation of the
dfdA1
promoter (P

dfdA1

) was dependent on
dfdR
and DF and not on a metabolite of the DF pathway. The cell extract of a
Rhodococcus
strain that heterologously expressed DfdR showed electrophoretic mobility shift (EMS) activity for the P

dfdA1

DNA fragment in a DF-dependent manner. In addition, P

dfdA1

activation and EMS activity were observed with hydrophobic aromatic compounds comprising two or more aromatic rings, suggesting that DfdR has broad effector molecule specificity for several hydrophobic aromatic compounds.
Geobacter sulfurreducens
Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase
Authors: Bralley Patricia , Cozad Madeline , Jones George H.
Published: October 2008

The genome of
Geobacter sulfurreducens
contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using
G. sulfurreducens
total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in
Escherichia coli
. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of
G. sulfurreducens
rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3′ tails. This is the first characterization of a bacterial system that expresses separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.
Cellodextrin and Laminaribiose ABC Transporters in
Clostridium thermocellum
Authors: Nataf Yakir , Yaron Sima , Stahl Frank , Lamed Raphael , Bayer Edward A. , Scheper Thomas-Helmut , Sonenshein Abraham L. , Shoham Yuval
Published: October 2008

Clostridium thermocellum
is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate.
C. thermocellum
can efficiently utilize only β-1,3 and β-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in
C. thermocellum
. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G
2
to G
5
), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G
3
), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of
C. thermocellum
, in which cellodextrins (G
3
to G
5
) are assimilated faster than cellobiose.
Pleiotropic Roles of Polyglycerolphosphate Synthase of Lipoteichoic Acid in Growth of
Staphylococcus aureus
Cells
Authors: Oku Yusuke , Kurokawa Kenji , Matsuo Miki , Yamada Sakuo , Lee Bok-Luel , Sekimizu Kazuhisa
Published: October 2008

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium
Staphylococcus aureus
. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient
S. aureus
Δ
ltaS
mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, Δ
ltaS
mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of Δ
ltaS
mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the Δ
ltaS
mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the Δ
ltaS
mutation was found to be synthetically lethal with the Δ
tagO
mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function.
Transcriptome Divergence and the Loss of Plasticity in
Bacillus subtilis
after 6,000 Generations of Evolution under Relaxed Selection for Sporulation
Authors: Maughan Heather , Birky C. William , Nicholson Wayne L.
Published: October 2008

We used microarrays to identify the causes of sporulation deficiencies in
Bacillus subtilis
after 6,000 generations of evolution. We found that sporulation loss did not result from large-scale deletions; therefore, it must have resulted from smaller indels and/or substitutions. Transcription patterns of one strain versus its ancestor showed that sporulation was not initiated and suggested that sporulation loss may be part of an overall decline in plasticity.