bluuebunny/crossref_metadata_2025_split huggingface dataset:
Aromatic Amino Acid Auxotrophs Constructed by Recombinant Marker Exchange in
Methylophilus methylotrophus
AS1 Cells Expressing the
aroP
-Encoded Transporter of
Escherichia coli
Authors: Yomantas Yurgis A. V. , Tokmakova Irina L. , Gorshkova Natalya V. , Abalakina Elena G. , Kazakova Svetlana M. , Gak Evgueni R. , Mashko Sergey V. 
Published: October 2009

ABSTRACT

The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium
Methylophilus methylotrophus
AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the
Escherichia coli
gene
aroP
, which encodes the common aromatic amino acid transporter, into the genome of
M. methylotrophus
. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp.
M. methylotrophus
AS1 genes
trpE
,
tyrA
,
pheA
, and
aroG
were cloned in
E. coli
, sequenced, disrupted in vitro using a Km
r
marker, and electroporated into an
aroP
carrier recipient strain. This approach led to the construction of a set of marker-less
M. methylotrophus
AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.
7-Ketocholesterol Catabolism by
Rhodococcus jostii
RHA1
Authors: Mathieu Jacques M. , Mohn William W. , Eltis Lindsay D. , LeBlanc Justin C. , Stewart Gord R. , Dresen Carola , Okamoto Kenji , Alvarez Pedro J. J. 
Published: October 2009

ABSTRACT

Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by
Rhodococcus jostii
RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.
Organization and Evolutionary Trajectory of the Mating Type (MAT) Locus in Dermatophyte and Dimorphic Fungal Pathogens
Authors: Li Wenjun , Metin Banu , White Theodore C. , Heitman Joseph 
Published: October 2009

ABSTRACTSexual reproduction in fungi is governed by a specialized genomic region, the mating type (MAT) locus, whose gene identity, organization, and complexity are diverse. We identified theMATlocus of five dermatophyte fungal pathogens (Microsporum gypseum,Microsporum canis,Trichophyton equinum,Trichophyton rubrum, andTrichophyton tonsurans) and a dimorphic fungus,Paracoccidioides brasiliensis, and performed phylogenetic analyses. The identifiedMATlocus idiomorphs ofM. gypseumcontrol cell type identity in mating assays, and recombinant progeny were produced. Virulence tests inGalleria mellonellalarvae suggest the two mating types ofM. gypseummay have equivalent virulence. Synteny analysis revealed common features of theMATlocus shared among these five dermatophytes: namely, a small size (∼3 kb) and a novel gene arrangement. TheSLA2,COX13, andAPN2genes, which flank theMATlocus in otherAscomycotaare instead linked on one side of the dermatophyteMATlocus. In addition, the transcriptional orientations of theAPN2andCOX13genes are reversed compared to the dimorphic fungiHistoplasma capsulatum,Coccidioides immitis, andCoccidioides posadasii. A putative transposable element,pogo, was found to have inserted in theMAT1-2idiomorph of oneP. brasiliensisstrain but not others. In conclusion, the evolution of theMATlocus of the dermatophytes and dimorphic fungi from the last common ancestor has been punctuated by both gene acquisition and expansion, and asymmetric gene loss. These studies further support a foundation to develop molecular and genetic tools for dermatophyte and dimorphic human fungal pathogens.
Campylobacter jejuni
FlpA Binds Fibronectin and Is Required for Maximal Host Cell Adherence
Authors: Konkel Michael E. , Larson Charles L. , Flanagan Rebecca C. 
Published: October 2009

ABSTRACT

Campylobacter jejuni
is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of
C. jejuni
to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a ∼250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (∼45 amino acids), type II (∼60 amino acids), and type III (∼90 amino acids). The deduced amino acid sequence of
C. jejuni flpA
(Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of
C. jejuni
to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in
C. jejuni
binding to host cells by in vitro adherence assays with a
C. jejuni
wild-type strain and a
C. jejuni flpA
mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the
C. jejuni flpA
mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked
C. jejuni
adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates
C. jejuni
attachment to host epithelial cells via Fn binding.
Maturation of 23S rRNA inBacillus subtilisin the Absence of Mini-III
Authors: Redko Yulia , Condon Ciarán 
Published: October 2009

ABSTRACT23S rRNA maturation inBacillus subtilisis catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5′-to-3′ exoribonuclease RNase J1 and the 3′-to-5′ exoribonucleases, principally RNase PH and YhaM.
Isolation of Basal Bodies with C-Ring Components from the Na
+
-Driven Flagellar Motor of
Vibrio alginolyticus
Authors: Koike Masafumi , Terashima Hiroyuki , Kojima Seiji , Homma Michio 
Published: October 2009

ABSTRACT

To investigate the Na
+
-driven flagellar motor of
Vibrio alginolyticus
, we attempted to isolate its C-ring structure. FliG but not FliM copurified with the basal bodies. FliM proteins may be easily dissociated from the basal body. We could detect FliG on the MS ring surface of the basal bodies.
HspR Mutations Are Naturally Selected in
Bifidobacterium longum
When Successive Heat Shock Treatments Are Applied
Authors: Berger B. , Moine D. , Mansourian R. , Arigoni F. 
Published: October 2009

ABSTRACT

The development of molecular tools allowed light to be shed on several widespread genetic mechanisms aiming at limiting the effect of molecular damage on bacterial survival. For some bacterial taxa, there are limited tools in the genetic toolbox, which restricts the possibilities to investigate the molecular basis of their stress response. In that case, an alternative strategy is to study genetic variants of a strain under stress conditions. The comparative study of the genetic determinants responsible for their phenotypes, e.g., an improved tolerance to stress, offers precious clues on the molecular mechanisms effective in this bacterial taxon. We applied this approach and isolated two heat shock-tolerant strains derived from
Bifidobacterium longum
NCC2705. A global analysis of their transcriptomes revealed that the
dnaK
operon and the
clpB
gene were overexpressed in both heat shock-tolerant strains. We sequenced the
hspR
gene coding for the negative regulator of
dnaK
and
clpB
and found point mutations affecting protein domains likely responsible for the binding of the regulators to the promoter DNA. Complementation of the mutant strains by the wild-type regulator
hspR
restored its heat sensitivity and thus demonstrated that these mutations were responsible for the observed heat tolerance phenotype.
Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme, Involved in the Late Steps of Coenzyme B
12
Biosynthesis in
Salmonella enterica
Authors: Claas Kathy R. , Parrish J. R. , Maggio-Hall L. A. , Escalante-Semerena J. C. 
Published: October 2009

ABSTRACT

In
Salmonella enterica
, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M
cobT
mutations were deleterious, we determined the nature of three class M
cobT
alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT
WT
) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT
WT
. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M
cobT
alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.
Linear Plasmid SLP2 Is Maintained by Partitioning, Intrahyphal Spread, and Conjugal Transfer inStreptomyces
Authors: Hsu Chin-Chen , Chen Carton W. 
Published: October 2009

ABSTRACTLow-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids inStreptomyces. SLP2 is a 50-kb low-copy-number linear plasmid inStreptomyces lividans, which contains a typicalparABpartitioning operon. InS. lividansandStreptomyces coelicolor, aparABdeletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-containing hyphae to plasmidless hyphae. Deletion of the transfer (traB) gene eliminated conjugal transfer, lessened the growth retardation of colonies, and increased plasmid loss through sporulation cycles. The additional deletion of an intrahyphal spread gene (spd1) caused almost complete plasmid loss in a sporulation cycle and eliminated all growth retardation. Moreover, deletion ofspd1alone severely reduced conjugal transfer and stability of SLP2 inS. coelicolorM145 but had no effect onS. lividansTK64. These results revealed the following three systems for SLP2 maintenance: partitioning and spread for moving the plasmid DNA along the hyphae and into spores and conjugal transfer for rescuing plasmidless hyphae. InS. lividans, both spread and partitioning appear to overlap functionally, but inS. coelicolor, spread appears to play the main role.
Discovery and Characterization of Three NewEscherichia coliSeptal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA
Authors: Arends S. J. Ryan , Williams Kyle , Scott Renada J. , Rolong Silvana , Popham David L. , Weiss David S. 
Published: October 2009

ABSTRACTSPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins fromEscherichia coliandCaulobacter crescentushave been shown to bind peptidoglycan. Besides FtsN,E. colihas three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring inE. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, includingAquifex aeolicus, localized to septal rings when produced inE. coliand bound to purifiedE. colipeptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.
A Type VI Secretion System Effector Protein, VgrG1, from
Aeromonas hydrophila
That Induces Host Cell Toxicity by ADP Ribosylation of Actin
Authors: Suarez G. , Sierra J. C. , Erova T. E. , Sha J. , Horneman A. J. , Chopra A. K. 
Published: October 2009

ABSTRACT

We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of
Aeromonas hydrophila
and showed that VasH, a σ
54
activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of
A. hydrophila
, namely, a Δ
act
mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Δ
act
Δ
vasH
mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the
vgrG1
gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and
Escherichia coli
, respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of
A. hydrophila
possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.
Competitive Inhibitions of the Chlorophyll Synthase of
Synechocystis
sp. Strain PCC 6803 by Bacteriochlorophyllide
a
and the Bacteriochlorophyll Synthase of
Rhodobacter sphaeroides
by Chlorophyllide
a
Authors: Kim Eui-Jin , Lee Jeong K. 
Published: October 2009

ABSTRACT

The photosynthetic growth of
Synechocystis
sp. strain PCC 6803 is hampered by exogenously added bacteriochlorophyllide
a
(Bchlide
a
) in a dose-dependent manner. The growth inhibition caused by Bchlide
a
, however, is relieved by an increased level of exogenously added chlorophyllide
a
(Chlide
a
). The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide
a
, with inhibition constants (
K
I
s) of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of
Rhodobacter sphaeroides
is inhibited competitively by Chlide
a
, with
K
I
s of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, exogenously added Chlide
a
inhibits the metabolic conversion of exogenously added Bchlide
a
to bacteriochlorophyll
a
by an
R. sphaeroides bchFNB-bchZ
mutant that neither synthesizes nor metabolizes Chlide
a
. The metabolic inhibition by Chlide
a
, however, is relieved by the elevated level of Bchlide
a
. Thus, the chlorophyll synthase of
Synechocystis
sp. PCC 6803 and the bacteriochlorophyll synthase of
R. sphaeroides
, both of which perform ping-pong-type reactions, are inhibited by Bchlide
a
and Chlide
a
, respectively. Although neither inhibitor is catalyzed by the target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites.
Computational fluid dynamic simulation to assess flow characteristics of an in vitro aneurysm model
Authors: Lott D A , Siegel M , Chaudhry H R , Prestigiacomo C J 
Published: October 2009

BackgroundModifications of in vitro aneurysm modeling to study the effects of morphology on flow dynamics are time consuming, costly and analysis tends to be more qualitative than quantitative. This study develops a virtual two-dimensional flow model replicating an in vitro aneurysm model and analyzes how changes in morphology modify flow characteristics.MethodsUsing finite volume analysis, a two-dimensional saccular aneurysm model was created with a configuration matching a published, experimental, in vitro model. Qualitative comparisons were made determining whether a two-dimensional fluid dynamic model can replicate the results of an in vitro model. Quantitative changes in flow patterns, wall shear stress, dynamic pressure and maximum velocities were assessed by modifying the shape of the neck and proximal dome without modifying the overall size of the aneurysm.ResultsA two-dimensional computational fluid dynamic model reproducing the shape of a published aneurysm demonstrated excellent qualitative fidelity to an in vitro flow model. Additional information regarding dynamic pressure, shear stress and velocity along the aneurysm neck and within the aneurysm dome were determined. Although all dimensions were kept constant, slight modifications of the neck and proximal dome resulted in quantitative changes in studied parameters, such as wall shear stress and dynamic pressure.ConclusionsComputer generated aneurysm flow models, when carefully developed, reproduce flow events within in vitro aneurysms providing objective data on biophysical parameters. Effective flow modeling of aneurysms depends on flow input, size of the parent vessel and aneurysm, and other factors. These data suggest that neck and proximal dome configuration, independent of size, are important characteristics of flow.
The impact of timing on outcomes of carotid artery stenting in recently symptomatic patients
Authors: Lin R , Mazighi M , Yadav J , Abou-Chebl A 
Published: October 2009

ObjectivesWe sought to determine whether the timing of carotid artery stenting (CAS) affects peri-procedural outcomes in recently symptomatic patients.BackgroundEarly carotid endarterectomy following a stroke lowers the risk of recurrent ischemic events but has been associated with an increased risk of intracerebral hemorrhage. The optimal timing of CAS following a stroke is unknown.MethodsUsing a single-center prospective CAS registry, we retrospectively analyzed data on consecutive CAS procedures over an 8-year period. Patients were separated into early (≤4 weeks) and late (>4 weeks) treatment groups based on time to CAS from symptom onset. Post-procedural hypertension was treated. Thirty-day incidence of stroke, intracerebral hemorrhage, myocardial infarction and death were recorded.ResultsA total of 224 patients were treated for symptomatic lesions. The mean age was 71±10 years, and 63% were men. The median time for CAS was 8 days (0.2–30 days) and 90 days (31–180 days) respectively, for the early (n=122) and late (n=102) intervention groups. There were 4 (3.45%) periprocedural strokes in the early and 5 (5.95%) in the late CAS group (p=0.5). There was one hyperperfusion syndrome in each group. The 30-day stroke, myocardial infarction or death rates were similar between the early (6.03%) and late (8.33%) CAS groups, p=0.58. A multivariate analysis showed that age >80 years rather than timing of CAS was associated with adverse events.ConclusionsCAS may be performed safely immediately following non-disabling strokes. Octogenarians may be at higher risk of complications.
The use of a covered stent graft for obliteration of high-flow carotid cavernous fistula presenting with life-threatening epistaxis
Authors: Kalia J S , Niu T , Zaidat O O 
Published: October 2009

BackgroundWe present a rare complication of trans-sphenoidal adenectomy (TSA) for pituitary macroadenoma: carotid cavernous fistula (CCF) that was treated with endovascular therapy. The incidence of internal carotid artery (ICA) injury following TSA is 1% and may spontaneously heal by packing and rarely manifest as symptomatic CCF/aneurysm. Treatment of post-TSA CCF may be challenging due to the breach of nasal floor and may be prone to recurrence.Presentation/interventionUncontrolled intra-operative bleeding during a TSA led to an emergent angiogram to show slow-flow left CCF. Due to clinical deterioration with nasal bleeding, angiography was repeated after 4 h; the fistula had transformed into high flow with significant increase in size, and was therefore embolized using stent-assisted coiling. The fistula recanalized in a month with massive epistaxis and was re-treated using a covered stent graft.ConclusionThis case represents several unique learning points: (1) CCF as a complication of TSA due to close anatomical proximity; (2) the role of endovascular management post-TSA complication; (3) stent-assisted coil embolization of high-flow fistula with moderate ICA laceration; (4) recanalization of CCF causing massive epistaxis; (5) rare use of covered stent graft stent in distal intracranial circulation maintaining integrity and patency of ICA; (6) long-term results after covered stent graft with no in-stent restenosis.