bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTCryptococcus 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

ABSTRACT

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

ABSTRACT

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

ABSTRACTEarly 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

ABSTRACTBacteriophage 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.