bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics and Biodiversity of Populations of Lactic Acid Bacteria andAcetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans inGhana
Authors: Camu Nicholas , De Winter Tom , Verbrugghe Kristof , Cleenwerck Ilse , Vandamme Peter , Takrama Jemmy S. , Vancanneyt Marc , De Vuyst Luc
Published: February 2007

The Ghanaian cocoa bean heap fermentation process was studied through a
multiphasic approach, encompassing both microbiological and metabolite
target analyses. A culture-dependent (plating and incubation, followed
by repetitive-sequence-based PCR analyses of picked-up
colonies) and culture-independent (denaturing gradient gel
electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach
revealed a limited biodiversity and targeted population dynamics of
both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during
fermentation. Four main clusters were identified among the LAB
isolated:
Lactobacillus plantarum
,
Lactobacillus
fermentum
,
Leuconostoc pseudomesenteroides
, and
Enterococcus casseliflavus
. Other taxa encompassed, for
instance,
Weissella
. Only four clusters were found
among the AAB identified:
Acetobacter pasteurianus
,
Acetobacter syzygii
-like bacteria, and two small clusters of
Acetobacter tropicalis
-like bacteria. Particular strains of
L. plantarum
,
L. fermentum
, and
A.
pasteurianus
, originating from the environment, were well adapted
to the environmental conditions prevailing during Ghanaian cocoa bean
heap fermentation and apparently played a significant role in the cocoa
bean fermentation process. Yeasts produced ethanol from
sugars, and LAB produced lactic acid, acetic acid, ethanol, and
mannitol from sugars and/or citrate. Whereas
L. plantarum
strains were abundant in the beginning of the fermentation,
L.
fermentum
strains converted fructose into mannitol upon prolonged
fermentation.
A. pasteurianus
grew on ethanol, mannitol, and
lactate and converted ethanol into acetic acid. A newly proposed
Weissella
sp., referred to as “
Weissella
ghanaensis
,” was detected through PCR-DGGE analysis in
some of the fermentations and was only occasionally picked up through
culture-based isolation. Two new species of
Acetobacter
were
found as well, namely, the species tentatively named“
Acetobacter senegalensis
” (
A.
tropicalis
-like) and “
Acetobacter
ghanaensis
” (
A.
syzygii
-like).
A Novel
lux
Operon in the Cryptically Bioluminescent Fish Pathogen
Vibrio salmonicida
Is Associated with Virulence
Authors: Nelson Eric J. , Tunsjø Hege S. , Fidopiastis Pat M. , Sørum Henning , Ruby Edward G.
Published: February 2007

The cold-water-fish pathogen
Vibrio salmonicida
expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the
lux
operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the
V. salmonicida lux
operon revealed that homologs of all of the genes required for luminescence are present:
luxAB
(luciferase) and
luxCDE
(aliphatic-aldehyde synthesis). The arrangement and sequence of these structural
lux
genes are conserved compared to those in related species of luminous bacteria. However,
V. salmonicida
strains have a novel arrangement and number of homologs of the
luxR
and
luxI
quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the
luxA
gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the
luxA
mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the
lux
operon. Nevertheless, these findings document for the first time an association between a mutation in a structural
lux
gene and virulence, as well as provide a new molecular system to study
Vibrio
pathogenesis in a natural host.
Tropheryma whipplei
in the Environment: Survey of Sewage Plant Influxes and Sewage Plant Workers
Authors: Schöniger-Hekele Maximilian , Petermann Dagmar , Weber Beate , Müller Christian
Published: February 2007

We studied the prevalence of
Tropheryma whipplei
in influxes to 46 sewage treatment plants and in stool, mouthwash fluids, and dental plaques of 64 healthy workers in those facilities and 146 disease control patients.
T. whipplei
was found in sewage water, in stool of healthy individuals, and significantly more often in stool of workers exposed to sewage water.
Use of High-Affinity Cell Wall-Binding Domains of Bacteriophage Endolysins for Immobilization and Separation of Bacterial Cells
Authors: Kretzer Jan W. , Lehmann Rainer , Schmelcher Mathias , Banz Manuel , Kim Kwang-Pyo , Korn Corinna , Loessner Martin J.
Published: February 2007

Immobilization and magnetic separation for specific enrichment of microbial cells, such as the pathogen
Listeria monocytogenes
, depends on the availability of suitable affinity molecules. We report here a novel concept for the immobilization and separation of bacterial cells by replacing antibodies with cell wall-binding domains (CBDs) of bacteriophage-encoded peptidoglycan hydrolases (endolysins). These polypeptide modules very specifically recognize and bind to ligands on the gram-positive cell wall with high affinity. With paramagnetic beads coated with recombinant
Listeria
phage endolysin-derived CBD molecules, more than 90% of the viable
L. monocytogenes
cells could be immobilized and recovered from diluted suspensions within 20 to 40 min. Recovery rates were similar for different species and serovars of
Listeria
and were not affected by the presence of other microorganisms. The CBD-based magnetic separation (CBD-MS) procedure was evaluated for capture and detection of
L. monocytogenes
from artificially and naturally contaminated food samples. The CBD separation method was shown to be superior to the established standard procedures; it required less time (48 h versus 96 h) and was the more sensitive method. Furthermore, the generalizability of the CBD-MS approach was demonstrated by using specific phage-encoded CBDs specifically recognizing
Bacillus cereus
and
Clostridium perfringens
cells, respectively. Altogether, CBD polypeptides represent novel and innovative tools for the binding and capture of bacterial cells, with many possible applications in microbiology and diagnostics.
Human Enteropathogen Load in Activated Sewage Sludge and Corresponding Sewage Sludge End Products
Authors: Graczyk Thaddeus K. , Lucy Frances E. , Tamang Leena , Miraflor Allen
Published: February 2007

This study demonstrated a significant reduction in the concentrations of
Cryptosporidium parvum
and
Cryptosporidium hominis
oocysts,
Giardia lamblia
cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.
The Antifungal Protein AFP from
Aspergillus giganteus
Inhibits Chitin Synthesis in Sensitive Fungi
Authors: Hagen Silke , Marx Florentine , Ram Arthur F. , Meyer Vera
Published: February 2007

The antifungal protein AFP from
Aspergillus giganteus
is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of
Fusarium oxysporum
and
Aspergillus oryzae
showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an
Aspergillus niger
reporter strain in which the cell wall integrity pathway was strongly induced by AFP.
Water Transport in Intact Yeast Cells as Assessed by Fluorescence Self-Quenching
Authors: Soveral Graça , Madeira Ana , Loureiro-Dias Maria C. , Moura Teresa F.
Published: February 2007

Intact yeast cells loaded with 5- and-6-carboxyfluorescein were used to assess water transport. The results were similar to those previously reported for protoplasts assessed by using either fluorescence or light scattering, and the activation energies were 8.0 and 15.1 kcal mol
−1
(33.4 and 63.2 kJ mol
−1
) for a strain overexpressing
AQY1
aquaporin and a parental strain, respectively.
Quantitative Real-Time PCR Detection of Toxic
Nodularia
Cyanobacteria in the Baltic Sea
Authors: Koskenniemi Kerttu , Lyra Christina , Rajaniemi-Wacklin Pirjo , Jokela Jouni , Sivonen Kaarina
Published: February 2007

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing
Nodularia
, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (
ndaF
), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30
ndaF
gene copies ml
−1
of seawater could be detected, and thus, the method was very sensitive. The
ndaF
gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by
Nodularia
. This qPCR method for the
ndaF
gene can be used for detailed studies of
Nodularia
blooms and their formation.
ndaF
gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic
Nodularia
blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin.
Developmental Regulation of an Adhesin Gene during Cellular Morphogenesis in the Fungal Pathogen
Candida albicans
Authors: Argimón Silvia , Wishart Jill A. , Leng Roger , Macaskill Susan , Mavor Abigail , Alexandris Thomas , Nicholls Susan , Knight Andrew W. , Enjalbert Brice , Walmsley Richard , Odds Frank C. , Gow Neil A. R. , Brown Alistair J. P.
Published: February 2007

Candida albicans
expresses specific virulence traits that promote disease establishment and progression. These traits include morphological transitions between yeast and hyphal growth forms that are thought to contribute to dissemination and invasion and cell surface adhesins that promote attachment to the host. Here, we describe the regulation of the adhesin gene
ALS3
, which is expressed specifically during hyphal development in
C. albicans
. Using a combination of reporter constructs and regulatory mutants, we show that this regulation is mediated by multiple factors at the transcriptional level. The analysis of
ALS3
promoter deletions revealed that this promoter contains two activation regions: one is essential for activation during hyphal development, while the second increases the amplitude of this activation. Further deletion analyses using the
Renilla reniformis
luciferase reporter delineate the essential activation region between positions −471 and −321 of the promoter. Further 5′ or 3′ deletions block activation.
ALS3
transcription is repressed mainly by Nrg1 and Tup1, but Rfg1 contributes to this repression. Efg1, Tec1, and Bcr1 are essential for the transcriptional activation of
ALS3
, with Tec1 mediating its effects indirectly through Bcr1 rather than through the putative Tec1 sites in the
ALS3
promoter.
ALS3
transcription is not affected by Cph2, but Cph1 contributes to full
ALS3
activation. The data suggest that multiple morphogenetic signaling pathways operate through the promoter of this adhesin gene to mediate its developmental regulation in this major fungal pathogen.
Streptococcus gordoniiHsa Environmentally Constrains Competitive Binding byStreptococcus sanguinisto Saliva-Coated Hydroxyapatite
Authors: Nobbs Angela H. , Zhang Yongshu , Khammanivong Ali , Herzberg Mark C.
Published: February 2007

Competition between pioneer colonizing bacteria may determine polymicrobial succession during dental plaque development, but the ecological constraints are poorly understood. For example, moreStreptococcus sanguinisthanStreptococcus gordoniiorganisms are consistently isolated from the same intraoral sites, yetS. gordoniifails to be excluded and survives as a species over time. To explain this observation, we hypothesized thatS. gordoniicould compete withS. sanguinisto adhere to saliva-coated hydroxyapatite (sHA), an in vitro model of the tooth surface. Both species bound similarly to sHA, yet 10- to 50-fold excessS. gordoniiDL1 reduced binding ofS. sanguinisSK36 by 85 to >95%.S. sanguinis, by contrast, did not significantly compete withS. gordoniito adhere.S. gordoniicompeted withS. sanguinismore effectively than other species of oral streptococci and depended upon the salivary film on HA. Next, putativeS. gordoniiadhesins were analyzed for contributions to interspecies competitive binding. Like wild-typeS. gordonii, isogenic mutants with mutations in antigen I/II polypeptides (sspAB), amylase-binding proteins (abpAB), and Csh adhesins (cshAB) competed effectively againstS. sanguinis. By contrast, anhsa-deficient mutant ofS. gordoniishowed significantly reduced binding and competitive capabilities, while these properties were restored in anhsa-complemented strain. Thus, Hsa confers a selective advantage toS. gordoniioverS. sanguinisin competitive binding to sHA. Hsa expression may, therefore, serve as an environmental constraint againstS. sanguinis, enablingS. gordoniito persist within the oral cavity, despite the greater natural prevalence ofS. sanguinisin plaque and saliva.
Species-Specific Functioning of the
Pseudomonas
XcpQ Secretin: Role for the C-Terminal Homology Domain and Lipopolysaccharide
Authors: Bitter Wilbert , van Boxtel Ria , Groeneweg Mathijs , Carballo Patricia Sánchez , Zähringer Ulrich , Tommassen Jan , Koster Margot
Published: February 2007

Secretins are oligomeric proteins that mediate the export of macromolecules across the bacterial outer membrane. The members of the secretin superfamily possess a C-terminal homology domain that is important for oligomerization and channel formation, while their N-terminal halves are thought to be involved in system-specific interactions. The XcpQ secretin of
Pseudomonas
spp. is a component of the type II secretion pathway. XcpQ from
Pseudomonas alcaligenes
is not able to functionally replace the secretin of the closely related species
Pseudomonas aeruginosa.
By analysis of chimeric XcpQ proteins, a region important for species-specific functioning was mapped between amino acid residues 344 and 478 in the C-terminal homology domain. Two chromosomal suppressor mutations were obtained that resulted in the proper functioning in
P. aeruginosa
of
P. alcaligenes
XcpQ and inactive hybrids. These mutations caused a defect in the synthesis of the lipopolysaccharide (LPS) outer core region. Subsequent analysis of different LPS mutants showed that changes in the outer core and not the loss of O antigen caused the suppressor phenotype. High concentrations of divalent cations in the growth medium also allowed
P. alcaligenes
XcpQ and inactive hybrids to function properly in
P. aeruginosa
. Since divalent cations are known to affect the structure of LPS, this observation supports the hypothesis that LPS has a role in the functioning of secretins.
A Phosphoenolpyruvate-Dependent Phosphotransferase System Is the Principal Maltose Transporter in
Streptococcus mutans
Authors: Webb Alexander J. , Homer Karen A. , Hosie Arthur H. F.
Published: February 2007

We report that a phosphoenolpyruvate-dependent phosphotransferase system, MalT, is the principal maltose transporter for
Streptococcus mutans.
MalT also contributes to maltotriose uptake. Since maltose and maltodextrins are products of starch degradation found in saliva, the ability to take up and ferment these carbohydrates may contribute to dental caries.
Performance of the Translational Apparatus Varies with the Ecological Strategies of Bacteria
Authors: Dethlefsen Les , Schmidt Thomas M.
Published: February 2007

Protein synthesis is the predominant activity of growing bacteria; the protein synthesis system accounts for more than one-half the cell's dry mass and consumes most of the cell's energy during rapid growth. Translation has been studied extensively using model organisms, and the translational apparatus is qualitatively similar in terms of structure and function across all known forms of life. However, little is known about variation between organisms in translational performance. Using measurements of macromolecular content in a phylogenetically diverse collection of bacteria with contrasting ecological strategies, we found that the translational power (the rate of protein synthesis normalized to the mass of the protein synthesis system) is three- to fourfold higher among bacteria that respond rapidly to nutrient availability than among bacteria that respond slowly. An analysis of codon use in completely sequenced bacterial genomes confirmed that the selective forces acting on translation vary with the ecological strategy. We propose that differences in translational power result from ecologically based variation among microbes in the relative importance of two competing benefits: reducing the biomass invested in the protein synthesis system and reducing the energetic expense of protein synthesis.
Intermolecular Forces and Enthalpies in the Adhesion of
Streptococcus mutans
and an Antigen I/II-Deficient Mutant to Laminin Films
Authors: Busscher Henk J. , van de Belt-Gritter Betsy , Dijkstra Rene J. B. , Norde Willem , Petersen Fernanda C. , Scheie Anne A. , van der Mei Henny C.
Published: February 2007

The antigen I/II family of surface proteins is expressed by most oral streptococci, including
Streptococcus mutans
, and mediates specific adhesion to, among other things, salivary films and extracellular matrix proteins. In this study we showed that antigen I/II-deficient
S. mutans
isogenic mutant IB03987 was nearly unable to adhere to laminin films under flow conditions due to a lack of specific interactions (0.8 × 10
6
and 1.1 × 10
6
cells cm
−2
at pH 5.8 and 6.8, respectively) compared with parent strain LT11 (21.8 × 10
6
and 26.1 × 10
6
cells cm
−2
). The adhesion of both the parent and mutant strains was slightly greater at pH 6.8 than at pH 5.8. In addition, atomic force microscopy (AFM) experiments demonstrated that the parent strain experienced less repulsion when it approached a laminin film than the mutant experienced. Upon retraction, combined specific and nonspecific adhesion forces were stronger for the parent strain (up to −5.0 and −4.9 nN at pH 5.8 and 6.8, respectively) than for the mutant (up to −1.5 and −2.1 nN), which was able to interact only through nonspecific interactions. Enthalpy was released upon adsorption of laminin to the surface of the parent strain but not upon adsorption of laminin to the surface of IB03987. A comparison of the adhesion forces in AFM with the adhesion forces reported for specific ligand-receptor complexes resulted in the conclusion that the number of antigen I/II binding sites for laminin on
S. mutans
LT11 is on the order of 6 × 10
4
sites per organism and that the sites are probably arranged along exterior surface structures, as visualized here by immunoelectron microscopy.
σ
B
Regulates IS
256
-Mediated
Staphylococcus aureus
Biofilm Phenotypic Variation
Authors: Valle Jaione , Vergara-Irigaray Marta , Merino Nekane , Penadés José R. , Lasa Iñigo
Published: February 2007

Biofilm formation in
Staphylococcus aureus
is subject to phase variation, and biofilm-negative derivatives emerge sporadically from a biofilm-positive bacterial population. To date, the only known mechanism for generating biofilm phenotypic variation in staphylococci is the reversible insertion/excision of IS
256
in biofilm-essential genes. In this study, we present evidence suggesting that the absence of the σ
B
transcription factor dramatically increases the rate of switching to the biofilm-negative phenotype in the clinical isolate
S. aureus
15981, under both steady-state and flow conditions. The phenotypic switching correlates with a dramatic increase in the number of IS
256
copies in the chromosomes of biofilm-negative variants, as well as with an augmented IS
256
insertion frequency into the
icaC
and the
sarA
genes. IS
256
-mediated biofilm switching is reversible, and biofilm-positive variants could emerge from biofilm-negative σ
B
mutants. Analysis of the chromosomal insertion frequency using a recombinant IS
256
element tagged with an erythromycin marker showed an almost three-times-higher transposition frequency in a Δσ
B
strain. However, regulation of IS
256
activity by σ
B
appears to be indirect, since transposase transcription is not affected in the absence of σ
B
and IS
256
activity is inhibited to wild-type levels in a Δσ
B
strain under NaCl stress. Overall, our results identify a new role for σ
B
as a negative regulator of insertion sequence transposition and support the idea that deregulation of IS
256
activity abrogates biofilm formation capacity in
S. aureus
.
Role of the PDZ Domains in
Escherichia coli
DegP Protein
Authors: Iwanczyk Jack , Damjanovic Daniela , Kooistra Joel , Leong Vivian , Jomaa Ahmad , Ghirlando Rodolfo , Ortega Joaquin
Published: February 2007

PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The
Escherichia coli
essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.
Genome of the Opportunistic Pathogen
Streptococcus sanguinis
Authors: Xu Ping , Alves Joao M. , Kitten Todd , Brown Arunsri , Chen Zhenming , Ozaki Luiz S. , Manque Patricio , Ge Xiuchun , Serrano Myrna G. , Puiu Daniela , Hendricks Stephanie , Wang Yingping , Chaplin Michael D. , Akan Doruk , Paik Sehmi , Peterson Darrell L. , Macrina Francis L. , Buck Gregory A.
Published: February 2007

The genome of
Streptococcus sanguinis
is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the
S. sanguinis
genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B
12
biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of
S. sanguinis
and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular,
S. sanguinis
possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.
The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams inStaphylococcus aureus
Authors: Truong-Bolduc Que Chi , Hooper David C.
Published: February 2007

MgrA is a known regulator of the expression of several multidrug transporters inStaphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression ofnorGproduces a threefold increase innorBtranscripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption ofnorGproduces no change in the level of transcripts ofnorA,norB, andnorCbut causes an increase of at least threefold in the transcript level ofabcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of clonedabcAcaused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects onnorBandabcAexpression. MgrA acts as an indirect repressor fornorBand a direct activator forabcA, whereas NorG acts as a direct activator fornorBand a direct repressor forabcA.
Characterization of the Signaling Domain of the NO-Responsive Regulator NorR from
Ralstonia eutropha
H16 by Site-Directed Mutagenesis
Authors: Klink Andrea , Elsner Bettina , Strube Katja , Cramm Rainer
Published: February 2007

In
Ralstonia eutropha
H16, the nitric oxide (NO)-responsive transcriptional activator NorR controls the expression of a dicistronic operon that encodes a membrane-bound NO reductase, NorB, and a protein of unknown function, NorA. The N-terminal domain (NTD) of NorR is responsible for perception of the signal molecule, nitric oxide. Thirteen out of 29 conserved residues of the NTD were exchanged by site-directed mutagenesis. Replacement of R63, R72, D93, D96, C112, D130, or F137 strongly decreased NorR-dependent promoter activation, while the exchange of Y95 or H110 led to an increase in promoter activity compared to that of the wild type. A purified truncated NorR comprising only the NTD (NorR-NTD) contained one iron atom per molecule and was able to bind NO in the as-isolated state. Based on the iron content of NorR-NTD proteins with single amino acid replacements, residues R72, D93, D96, C112, and D130 are likely candidates for iron ligands. Residues R63, Y95, and H110 appear not to be involved in NO binding but may take part in subsequent steps of the signal transduction mechanism of NorR.
Coordinated High-Light Response of Genes Encoding Subunits of Photosystem I Is Achieved by AT-Rich Upstream Sequences in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Muramatsu Masayuki , Hihara Yukako
Published: February 2007

Genes encoding subunits of photosystem I (PSI genes) in the cyanobacterium
Synechocystis
sp. strain PCC 6803 are actively transcribed under low-light conditions, whereas their transcription is coordinately and rapidly down-regulated upon the shift to high-light conditions. In order to identify the molecular mechanism of the coordinated high-light response, we searched for common light-responsive elements in the promoter region of PSI genes. First, the precise architecture of the
psaD
promoter was determined and compared with the previously identified structure of the
psaAB
promoter. One of two promoters of the
psaAB
genes (P1) and of the
psaD
gene (P2) possessed an AT-rich light-responsive element located just upstream of the basal promoter region. These sequences enhanced the basal promoter activity under low-light conditions, and their activity was transiently suppressed upon the shift to high-light conditions. Subsequent analysis of
psaC
,
psaE
,
psaK1
, and
psaLI
promoters revealed that their light response was also achieved by AT-rich sequences located at the −70 to −46 region. These results clearly show that AT-rich upstream elements are responsible for the coordinated high-light response of PSI genes dispersed throughout
Synechocystis
genome.
Region-Specific Insertion of Transposons in Combination with Selection for High Plasmid Transferability and Stability Accounts for the Structural Similarity of IncP-1 Plasmids
Authors: Sota Masahiro , Tsuda Masataka , Yano Hirokazu , Suzuki Haruo , Forney Larry J. , Top Eva M.
Published: February 2007

The overall architecture of IncP-1 plasmids is very conserved in that the accessory genes are typically located in one or two specific regions: betweenoriVandtrfAand between thetraandtrboperons. Various hypotheses have been formulated to explain this, but none have been tested experimentally. We investigated whether this structural similarity is due to region-specific transposition alone or also is reliant on selection for plasmids with insertions limited to these two regions. We first examined the transposition of Tn21Km into IncP-1β plasmid pBP136 and found that most Tn21Km insertions (67%) were located aroundoriV. A similar experiment using theoriVregion of IncP-1β plasmid pUO1 confirmed these results. We then tested the transferability, stability, and fitness cost of different pBP136 derivatives to determine if impairment of these key plasmid characters explained the conserved plasmid architecture. Most of the pBP136 derivatives with insertions in transfer genes were no longer transferable. The plasmids with insertions in theoriV-trfAandtra-trbregions were more stable than other plasmid variants, and one of these also showed a significantly lower fitness cost. In addition, our detailed sequence analysis of IncP-1 plasmids showed that Tn402/5053-like transposons are situated predominantly between thetraandtrboperons and close to the putative resolution site for the ParA resolvase, a potential hot spot for those transposons. Our study presents the first empirical evidence that region-specific insertion of transposons in combination with selection for transferable and stable plasmids explains the structural similarity of IncP-1 plasmids.
In Vivo Demonstration of FNR Dimers in Response to Lower O
2
Availability
Authors: Jervis Adrian J. , Green Jeffrey
Published: February 2007

Escherichia coli
FNR is an O
2
-sensing transcription factor. In vitro studies indicate that anaerobic iron-sulfur cluster acquisition promotes FNR dimerization. Here, two-hybrid assays show that iron-sulfur cluster-dependent FNR dimers are formed in vivo in response to lower O
2
availability, consistent with the current model of FNR activation.
His20Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain
Authors: Larsen Ray A. , Deckert Gail E. , Kastead Kyle A. , Devanathan Surendranathan , Keller Kimberly L. , Postle Kathleen
Published: February 2007

The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane ofEscherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His20is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser16and His20, the resultant “all-Ala Ser16His20” TMD TonB retained 90% of wild-type iron transport activity. Ser16Ala in the context of a wild-type TonB TMD was fully active. In contrast, His20Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser16His20TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser16His20TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser16His20TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser16His20TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.
Sortase-Catalyzed Assembly of Distinct Heteromeric Fimbriae in
Actinomyces naeslundii
Authors: Mishra Arunima , Das Asis , Cisar John O. , Ton-That Hung
Published: February 2007

Two types of adhesive fimbriae are expressed by
Actinomyces
; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of
Actinomyces naeslundii
strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the
fimQ
-
fimP
-
srtC1
-
fimR
gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the
fimB
-
fimA
-
srtC2
gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in
Actinomyces
and their function in the pathogenesis of different biofilm-related oral diseases.
Short-term pretreatment with low-dose hydrogen peroxide enhances the efficacy of bone marrow cells for therapeutic angiogenesis
Authors: Kubo Masayuki , Li Tao-Sheng , Suzuki Ryo , Ohshima Mako , Qin Shu-Lan , Hamano Kimikazu
Published: February 2007

Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). Hydrogen peroxide (H2O2) has been shown to increase VEGF expression and to be involved in angiogenesis. We tested the hypothesis that pretreatment with H2O2 enhances the efficacy of BMCs for neovascularization. H2O2 pretreatment was done by incubating mouse BMCs in 5 μM H2O2 for 30 min, followed by washing twice with PBS. The H2O2-pretreated and untreated BMCs were then studied in vitro and in vivo. RT-PCR analysis showed that expression of VEGF and Flk-1 mRNA was significantly higher in H2O2-pretreated BMCs than in untreated BMCs after 12 and 24 h of culture ( P < 0.01). Pretreatment with H2O2 also effectively enhanced the VEGF production and endothelial differentiation from BMCs after 1 and 7 days of culture ( P < 0.05). To estimate the angiogenic potency in vivo, H2O2-pretreated or untreated BMCs were intramuscularly implanted into the ischemic hindlimbs of mice. After 14 days of treatment, many of the H2O2-pretreated BMCs were viable, showed endothelial differentiation, and were incorporated in microvessels. Conversely, the survival and incorporation of the untreated BMCs were relatively poor. Microvessel density and blood flow in the ischemic hindlimbs were significantly greater in the mice implanted with H2O2-pretreated BMCs than in those implanted with untreated BMCs ( P < 0.05). These results show that the short-term pretreatment of BMCs with low-dose H2O2 is a novel, simple, and feasible method of enhancing their angiogenic potency.
Redox regulation of ischemic preconditioning is mediated by the differential activation of caveolins and their association with eNOS and GLUT-4
Authors: Koneru Srikanth , Penumathsa Suresh Varma , Thirunavukkarasu Mahesh , Samuel Samson Mathews , Zhan Lijun , Han Zhihua , Maulik Gautam , Das Dipak K. , Maulik Nilanjana
Published: February 2007

Reactive oxygen species (ROS) generated during ischemia-reperfusion (I/R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion [ischemic preconditioning (IP)] induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of GLUT-4 from the intracellular vesicles to the sarcolemma. In the present study we demonstrated involvement of ROS in IP-mediated GLUT-4 translocation along with increased expression of caveolin (Cav)-3, phospho (p)-endothelial nitric oxide synthase (eNOS), p-Akt, and decreased expression of Cav-1. The rats were divided into the following groups: 1) control sham, 2) N-acetyl-l-cysteine (NAC, free radical scavenger) sham (NS), 3) I/R, 4) IP + I/R (IP), and 5) NAC + IP (IPN). IP was performed by four cycles of 4 min of ischemia and 4 min of reperfusion followed by 30 min of ischemia and 3, 24, 48 h of reperfusion, depending on the protocol. Increased mRNA expression of GLUT-4 and Cav-3 was observed after 3 h of reperfusion in the IP group compared with other groups. IP increased expression of GLUT-4, Cav-3, and p-AKT and p-eNOS compared with I/R. Coimmunoprecipitation demonstrated decreased association of Cav-1/eNOS in the IP group compared with the I/R group. Significant GLUT-4 and Cav-3 association was also observed in the IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents a significant role of ROS signaling in Akt/eNOS/Cav-3-mediated GLUT-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP-induced eNOS and GLUT-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia, leading to myocardial protection in a clinically relevant rat ischemic model.
PTEN reduces cuff-induced neointima formation and proinflammatory cytokines
Authors: Koide Shinichiro , Okazaki Masahiro , Tamura Masahito , Ozumi Kiyoshi , Takatsu Hiroyuki , Kamezaki Fumihiko , Tanimoto Akihide , Tasaki Hiromi , Sasaguri Yasuyuki , Nakashima Yasuhide , Otsuji Yutaka
Published: February 2007

An inflammatory response followed by vascular injury plays an important role in neointima formation and development of atherosclerotic lesions, which are in part mediated by proinflammatory cytokines. Using a cuff injury model, we examined the effects of adenovirus-mediated overexpression of phosphatase and tensin homology deleted on chromosome 10 (PTEN) on neointima formation and the proinflammatory response. A cuff was placed around the femoral artery, and adenovirus expressing human PTEN type 1 (AdPTEN) or Escherichia coli β-galactosidase (AdLacZ) was injected between the cuff and the adventitia. After 14 days, the arteries were examined histopathologically and by Western blotting. The significant reduction of neointima formation by AdPTEN compared with AdLacZ was accompanied by reduced cell proliferation and increased adventitial cell apoptosis. AdPTEN also reduced expression of phosphorylated IκB-α, but not nonphosphorylated IκB-α. Western blotting revealed that AdPTEN reduced the cuff injury-induced expression levels of monocyte chemoattractant protein-1, TNF-α, and IL-1β and their expression in all layers of the arterial wall. In contrast, cuff-induced macrophage invasion, which was also inhibited by AdPTEN, was detected only at the intimal surface and in the adventitia. In cultured vascular smooth muscle cells, PTEN directly inhibited ANG II-induced monocyte chemoattractant protein-1 expression as quantified by real-time PCR and Western blotting. Our results suggest that overexpression of PTEN reduces neointima formation, possibly in part through inhibition of the inflammatory response by macrophage invasion and proinflammatory cytokine expression.
Effects of transmural pressure and wall shear stress on LDL accumulation in the arterial wall: a numerical study using a multilayered model
Authors: Sun Nanfeng , Wood Nigel B. , Hughes Alun D. , Thom Simon A. M. , Yun Xu X.
Published: February 2007

The accumulation of low-density lipoprotein (LDL) is recognized as one of the main contributors in atherogenesis. Mathematical models have been constructed to simulate mass transport in large arteries and the consequent lipid accumulation in the arterial wall. The objective of this study was to investigate the influences of wall shear stress and transmural pressure on LDL accumulation in the arterial wall by a multilayered, coupled lumen-wall model. The model employs the Navier-Stokes equations and Darcy's Law for fluid dynamics, convection-diffusion-reaction equations for mass balance, and Kedem-Katchalsky equations for interfacial coupling. To determine physiologically realistic model parameters, an optimization approach that searches optimal parameters based on experimental data was developed. Two sets of model parameters corresponding to different transmural pressures were found by the optimization approach using experimental data in the literature. Furthermore, a shear-dependent hydraulic conductivity relation reported previously was adopted. The integrated multilayered model was applied to an axisymmetric stenosis simulating an idealized, mildly stenosed coronary artery. The results show that low wall shear stress leads to focal LDL accumulation by weakening the convective clearance effect of transmural flow, whereas high transmural pressure, associated with hypertension, leads to global elevation of LDL concentration in the arterial wall by facilitating the passage of LDL through wall layers.
The role of the renin-angiotensin system and oxidative stress in spontaneously hypertensive rat mesenteric collateral growth impairment
Authors: Miller Steven J. , Norton Laura E. , Murphy Michael P. , Dalsing Michael C. , Unthank Joseph L.
Published: February 2007

Recent clinical and animal studies have shown that collateral artery growth is impaired in the presence of vascular risk factors, including hypertension. Available evidence suggests that angiotensin-converting enzyme inhibitors (ACEI) promote collateral growth in both hypertensive humans and animals; however, the specific mechanisms are not established. This study evaluated the hypothesis that collateral growth impairment in hypertension is mediated by excess superoxide produced by NAD(P)H oxidase in response to stimulation of the ANG II type 1 receptor. After ileal artery ligation, mesenteric collateral growth did not occur in untreated, young, spontaneously hypertensive rats. Significant luminal expansion occurred in collaterals of spontaneously hypertensive rats treated with the superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and the ACEI captopril, but not ANG II type 1 (losartan) or type 2 (PD-123319) receptor blockers. The ACEI enalapril produced equivalent reduction of arterial pressure as captopril but did not promote luminal expansion. This suggests the effects of captopril on collateral growth might result from its antioxidant properties. RT-PCR demonstrated that ANG II type 1 receptor and angiotensinogen expression was reduced in collaterals of untreated rats. This local suppression of the renin angiotensin system provides a potential explanation for the lack of effect of enalapril and losartan on collateral growth. The results demonstrate the capability of antioxidant therapies, including captopril, to reverse impaired collateral artery growth and the novel finding that components of the local renin angiotensin system are naturally suppressed in collaterals.
A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells
Authors: Inglis Sarah K. , Brown Sean G. , Constable Maree J. , McTavish Niall , Olver Richard E. , Wilson Stuart M.
Published: February 2007

By analysis of whole cell membrane currents in Na+-absorbing H441 human airway epithelial cells, we have identified a K+ conductance ( GK) resistant to Ba2+ but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K+ current ( IBl) whereas Ba2+ has only a weak inhibitory effect. IBl was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in IBl, acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K+ channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current ( ISC), and basolateral bupivacaine, lidocaine, quinidine, and Ba2+ inhibited ISC at concentrations similar to those needed to inhibit IBl, suggesting that the K+ channels underlying IBl are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K+ (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie GK in unstimulated cells and so maintain the driving force for Na+ absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.