bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptation and Response of
Bifidobacterium animalis
subsp.
lactis
to Bile: a Proteomic and Physiological Approach
Authors: Sánchez Borja , Champomier-Vergès Marie-Christine , Stuer-Lauridsen Birgitte , Ruas-Madiedo Patricia , Anglade Patricia , Baraige Fabienne , de los Reyes-Gavilán Clara G. , Johansen Eric , Zagorec Monique , Margolles Abelardo
Published: September 2007

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche.
Bifidobacterium animalis
subsp.
lactis
, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of
B. animalis
subsp.
lactis
IPLA 4549 and its bile-resistant derivative
B. animalis
subsp.
lactis
4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in
B. animalis
subsp.
lactis
. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in
B. animalis
subsp.
lactis
. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in
B. animalis
subsp.
lactis
involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.
Indirect Oxidation of Co(II) in the Presence of the Marine Mn(II)-Oxidizing Bacterium
Bacillus
sp. Strain SG-1
Authors: Murray Karen J. , Webb Samuel M. , Bargar John R. , Tebo Bradley M.
Published: September 2007

Cobalt(II) oxidation in aquatic environments has been shown to be linked to Mn(II) oxidation, a process primarily mediated by bacteria. This work examines the oxidation of Co(II) by the spore-forming marine Mn(II)-oxidizing bacterium
Bacillus
sp. strain SG-1, which enzymatically catalyzes the formation of reactive nanoparticulate Mn(IV) oxides. Preparations of these spores were incubated with radiotracers and various amounts of Co(II) and Mn(II), and the rates of Mn(II) and Co(II) oxidation were measured. Inhibition of Mn(II) oxidation by Co(II) and inhibition of Co(II) oxidation by Mn(II) were both found to be competitive. However, from both radiotracer experiments and X-ray spectroscopic measurements, no Co(II) oxidation occurred in the complete absence of Mn(II), suggesting that the Co(II) oxidation observed in these cultures is indirect and that a previous report of enzymatic Co(II) oxidation may have been due to very low levels of contaminating Mn. Our results indicate that the mechanism by which SG-1 oxidizes Co(II) is through the production of the reactive nanoparticulate Mn oxide.
Maximizing Recovery and Detection of
Cryptosporidium parvum
Oocysts from Spiked Eastern Oyster (
Crassostrea virginica
) Tissue Samples
Authors: Downey Autumn S. , Graczyk Thaddeus K.
Published: September 2007

Numerous studies have documented the presence of
Cryptosporidium parvum
, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of
Cryptosporidium
contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate
Cryptosporidium parvum
oocysts from oysters (
Crassostrea virginica
) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (
P
< 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with
Cryptosporidium
contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.
Degradation of Carbazole by Microbial Cells Immobilized in Magnetic Gellan Gum Gel Beads
Authors: Wang Xia , Gai Zhonghui , Yu Bo , Feng Jinhui , Xu Changyong , Yuan Yong , Lin Zhixin , Xu Ping
Published: September 2007

Polycyclic aromatic heterocycles, such as carbazole, are environmental contaminants suspected of posing human health risks. In this study, we investigated the degradation of carbazole by immobilized
Sphingomonas
sp. strain XLDN2-5 cells. Four kinds of polymers were evaluated as immobilization supports for
Sphingomonas
sp. strain XLDN2-5. After comparison with agar, alginate, and κ-carrageenan, gellan gum was selected as the optimal immobilization support. Furthermore, Fe
3
O
4
nanoparticles were prepared by a coprecipitation method, and the average particle size was about 20 nm with 49.65-electromagnetic-unit (emu) g
−1
saturation magnetization. When the mixture of gellan gel and the Fe
3
O
4
nanoparticles served as an immobilization support, the magnetically immobilized cells were prepared by an ionotropic method. The biodegradation experiments were carried out by employing free cells, nonmagnetically immobilized cells, and magnetically immobilized cells in aqueous phase. The results showed that the magnetically immobilized cells presented higher carbazole biodegradation activity than nonmagnetically immobilized cells and free cells. The highest biodegradation activity was obtained when the concentration of Fe
3
O
4
nanoparticles was 9 mg ml
−1
and the saturation magnetization of magnetically immobilized cells was 11.08 emu g
−1
. Additionally, the recycling experiments demonstrated that the degradation activity of magnetically immobilized cells increased gradually during the eight recycles. These results support developing efficient biocatalysts using magnetically immobilized cells and provide a promising technique for improving biocatalysts used in the biodegradation of not only carbazole, but also other hazardous organic compounds.
Antimicrobial Susceptibilities of
Vibrio parahaemolyticus
and
Vibrio vulnificus
Isolates from Louisiana Gulf and Retail Raw Oysters
Authors: Han Feifei , Walker Robert D. , Janes Marlene E. , Prinyawiwatkul Witoon , Ge Beilei
Published: September 2007

The antimicrobial susceptibilities of 168
Vibrio parahaemolyticus
and 151
Vibrio vulnificus
isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in
V. parahaemolyticus
for ampicillin (81%; MIC ≥ 16 μg/ml). Additionally,
V. parahaemolyticus
displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than
V. vulnificus
.
Identification and Expression Analyses of Putative Sesquiterpene Synthase Genes in
Phormidium
sp. and Prevalence of
geoA
-Like Genes in a Drinking Water Reservoir
Authors: Ludwig Frank , Medger Anja , Börnick Hilmar , Opitz Michael , Lang Kathrin , Göttfert Michael , Röske Isolde
Published: September 2007

The occurrence of taste and odor problems in drinking water supplies is a widespread phenomenon. From a Saxonian water reservoir we isolated a cyanobacterial species which was classified as
Phormidium
sp. Under laboratory conditions it produced an earthy-musty smell due to the synthesis of geosmin. The only genes shown to be involved in geosmin biosynthesis are
cyc2
and
geoA
of
Streptomyces
. Based on the alignment of Cyc2 with a putative sesquiterpene synthase of
Nostoc punctiforme
, a degenerate primer pair was designed. By PCR, we could amplify two similar genes in
Phormidium
sp., which we named
geoA1
and
geoA2
. Their expression was studied by reverse transcription-PCR. This revealed that both genes are expressed at 20°C and a light-dark cycle of 12 h. Expression was not detectable at the end of a 24-h dark period. To analyze the prevalence of
geoA1
and
geoA2
in samples from the phytobenthos, we generated PCR fragments with the same degenerate primer pair. Fifty-five different sequences that might represent
geoA
variants were obtained. The GC content ranged from 42% to 67%, suggesting that taxonomically very different bacteria might contain such genes.
Detection and Quantification of
Geobacter lovleyi
Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites
Authors: Amos Benjamin K. , Sung Youlboong , Fletcher Kelly E. , Gentry Terry J. , Wu Wei-Min , Criddle Craig S. , Zhou Jizhong , Löffler Frank E.
Published: September 2007

Geobacter lovleyi
strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing
Geobacter
group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related
Geobacter
isolates, including its closest cultured relative,
G. thiogenes
. Detection limits for direct and nested PCR were approximately 1 × 10
6
and 1 × 10
4
16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from
G. thiogenes
when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (
G. thiogenes
) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 10
7
± 0.1 × 10
7
per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The
G. lovleyi
strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.
Characterization of Regulatory Pathways in
Xylella fastidiosa
: Genes and Phenotypes Controlled by
algU
Authors: Shi Xiang Yang , Dumenyo C. Korsi , Hernandez-Martinez Rufina , Azad Hamid , Cooksey Donald A.
Published: September 2007

Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene
algU
, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in
Pseudomonas aeruginosa
and many other bacteria, has been extensively studied. The role of
algU
in
Xylella fastidiosa
, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an
algU
::
nptII
mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the
algU
::
nptII
mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.
Impact of Inorganic Carbon Availability on Microcystin Production by
Microcystis aeruginosa
PCC 7806
Authors: Jähnichen Sabine , Ihle Tilo , Petzoldt Thomas , Benndorf Jürgen
Published: September 2007

Batch culture experiments with the cyanobacterium
Microcystis aeruginosa
PCC 7806 were performed in order to test the hypothesis that microcystins (MCYSTs) are produced in response to a relative deficiency of intracellular inorganic carbon (C
i,i
). In the first experiment, MCYST production was studied under increased C
i,i
deficiency conditions, achieved by restricting sodium-dependent bicarbonate uptake through replacement of sodium bicarbonate in the medium with its potassium analog. The same experimental approach was used in a second experiment to compare the response of the wild-type strain
M. aeruginosa
PCC 7806 with its
mcyB
mutant, which lacks the ability to produce MCYSTs. In a third experiment, the impact of varying the C
i,i
status on MCYST production was examined without suppressing the sodium-dependent bicarbonate transporter; instead, a detailed investigation of a dark-light cycle was performed. In all experiments, a relative C
i,i
deficiency was indicated by an elevated variable fluorescence signal and led to enhanced phycocyanin cell quotas. Higher MCYST cell quotas (in the first and third experiments) and increased total (intracellular plus extracellular) MCYST production (in the first experiment) were detected with increased C
i,i
deficiency. Furthermore, the MCYST-producing wild-type strain and its
mcyB
mutant showed basically the same response to restrained inorganic carbon uptake, with elevated variable fluorescence and phycocyanin cell quotas with increased C
i,i
deficiency. The response of the wild type, however, was distinctly stronger and also included elevated chlorophyll
a
cell quotas. These differences indicate the limited ability of the mutant to adapt to low-C
i,i
conditions. We concluded that MCYSTs may be involved in enhancing the efficiency of the adaptation of the photosynthetic apparatus to fluctuating inorganic carbon conditions in cyanobacterial cells.
Impact of Environmental and Genetic Factors on Biofilm Formation by the Probiotic Strain
Lactobacillus rhamnosus
GG
Authors: Lebeer Sarah , Verhoeven Tine L. A. , Perea Vélez Mónica , Vanderleyden Jos , De Keersmaecker Sigrid C. J.
Published: September 2007

Lactobacillus rhamnosus
GG (ATCC 53103) is one of the clinically best-studied probiotic organisms. Moreover,
L. rhamnosus
GG displays very good in vitro adherence to epithelial cells and mucus. Here, we report that
L. rhamnosus
GG is able to form biofilms on abiotic surfaces, in contrast to other strains of the
Lactobacillus casei
group tested under the same conditions. Microtiter plate biofilm assays indicated that in vitro biofilm formation by
L. rhamnosus
GG is strongly modulated by culture medium factors and conditions related to the gastrointestinal environment, including low pH; high osmolarity; and the presence of bile, mucins, and nondigestible polysaccharides. Additionally, phenotypic analysis of mutants affected in exopolysaccharides (
wzb
), lipoteichoic acid (
dltD
), and central metabolism (
luxS
) showed their relative importance in biofilm formation by
L. rhamnosus
GG.
Molecular Characterization of Diazotrophic and Denitrifying Bacteria Associated with Mangrove Roots
Authors: Flores-Mireles Ana L. , Winans Stephen C. , Holguin Gina
Published: September 2007

An analysis of the molecular diversity of N
2
fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of
nifH
(N
2
fixation) and
nirS
and
nirK
(denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for
nifH
was higher than that for
nirK
or
nirS
at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest
nifH
diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For
nirS
, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for
nifH
. OTU defined on the basis of
nirK
were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of
nifH
,
nirK
, and
nirS
showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.
Cryptosporidium parvum
Long-Chain Fatty Acid Elongase
Authors: Fritzler Jason M. , Millership Jason J. , Zhu Guan
Published: September 2007

We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in
Cryptosporidium
and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. Cp
LCE1
gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C
14:0
) and palmitoyl-CoA (C
16:0
) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C
16:0
and C
14:0
were used as substrates, respectively). These observations ultimately validate the function of CpLCE1.
New Insights into the WalK/WalR (YycG/YycF) Essential Signal Transduction Pathway Reveal a Major Role in Controlling Cell Wall Metabolism and Biofilm Formation inStaphylococcus aureus
Authors: Dubrac Sarah , Boneca Ivo Gomperts , Poupel Olivier , Msadek Tarek
Published: September 2007

The highly conserved WalK/WalR (also known as YycG/YycF) two-component system is specific to low-G+C gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role have remained mostly uncharacterized. We observed that, unexpectedly,Staphylococcus aureuscell death induced by WalKR depletion was not followed by lysis. We show that WalKR positively controls autolytic activity, in particular that of the two majorS. aureusautolysins, AtlA and LytM. By using our previously characterized consensus WalR binding site and carefully reexamining the genome annotations, we identified nine genes potentially belonging to the WalKR regulon that appeared to be involved inS. aureuscell wall degradation. Expression of all of these genes was positively controlled by WalKR levels in the cell, leading to high resistance to Triton X-100-induced lysis when the cells were starved for WalKR. Cells lacking WalKR were also more resistant to lysostaphin-induced lysis, suggesting modifications in cell wall structure. Indeed, lowered levels of WalKR led to a significant decrease in peptidoglycan biosynthesis and turnover and to cell wall modifications, which included increased peptidoglycan cross-linking and glycan chain length. We also demonstrated a direct relationship between WalKR levels and the ability to form biofilms. This is the first example inS. aureusof a regulatory system positively controlling autolysin synthesis and biofilm formation. Taken together, our results now define this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR to reflect its true function.
Genome Sequence Analysis of the Emerging Human Pathogenic Acetic Acid Bacterium
Granulibacter bethesdensis
Authors: Greenberg David E. , Porcella Stephen F. , Zelazny Adrian M. , Virtaneva Kimmo , Sturdevant Dan E. , Kupko John J. , Barbian Kent D. , Babar Amenah , Dorward David W. , Holland Steven M.
Published: September 2007

Chronic granulomatous disease (CGD) is an inherited immune deficiency characterized by increased susceptibility to infection with
Staphylococcus
, certain gram-negative bacteria, and fungi.
Granulibacter bethesdensis
, a newly described genus and species within the family
Acetobacteraceae
, was recently isolated from four CGD patients residing in geographically distinct locales who presented with fever and lymphadenitis. We sequenced the genome of the reference strain of
Granulibacter bethesdensis
, which was isolated from lymph nodes of the original patient. The genome contains 2,708,355 base pairs in a single circular chromosome, in which 2,437 putative open reading frames (ORFs) were identified, 1,470 of which share sequence similarity with ORFs in the nonpathogenic but related
Gluconobacter oxydans
genome. Included in the 967 ORFs that are unique to
G. bethesdensis
are ORFs potentially important for virulence, adherence, DNA uptake, and methanol utilization. GC% values and best BLAST analysis suggested that some of these unique ORFs were recently acquired. Comparison of
G. bethesdensis
to other known CGD pathogens demonstrated conservation of some putative virulence factors, suggesting possible common mechanisms involved in pathogenesis in CGD. Genotyping of the four patient isolates by use of a custom microarray demonstrated genome-wide variations in regions encoding DNA uptake systems and transcriptional regulators and in hypothetical ORFs.
G. bethesdensis
is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.
Pseudomonas syringae
Lytic Transglycosylases Coregulated with the Type III Secretion System Contribute to the Translocation of Effector Proteins into Plant Cells
Authors: Oh Hye-Sook , Kvitko Brian H. , Morello Joanne E. , Collmer Alan
Published: September 2007

Pseudomonas syringae
translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by
hrp
(for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in
P. syringae
pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated
hrpH
), PSPTO2678 (
hopP1
), and PSPTO852 (
hopAJ1
).
hrpH
is located between
hrpR
and
avrE1
in the Hrp pathogenicity island and is carried in the functional cluster of
P. syringae
pv. syringae 61
hrp
genes cloned in cosmid pHIR11. Strong expression of DC3000
hrpH
in
Escherichia coli
inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (
Bordetella pertussis
adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates.
Pseudomonas fluorescens
carrying a pHIR11 derivative lacking
hrpH
is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH
1-241
. DC3000 mutants lacking
hrpH
or
hrpH, hopP1
, and
hopAJ1
combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH
1-241
variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection.
Polyphosphate Kinase 1 Is a Pathogenesis Determinant in
Campylobacter jejuni
Authors: Candon Heather L. , Allan Brenda J. , Fraley Cresson D. , Gaynor Erin C.
Published: September 2007

Campylobacter jejuni
is the leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, relatively little is known about
C. jejuni
's precise pathogenesis mechanisms, particularly in comparison to other well-studied enteric organisms such as
Escherichia coli
and
Salmonella
spp. Altered expression of phosphate genes in a
C. jejuni
stringent response mutant, together with known correlations between the stringent response, polyphosphate (poly-P), and virulence in other bacteria, led us to investigate the role of poly-P in
C. jejuni
stress survival and pathogenesis. All sequenced
C. jejuni
strains harbor a conserved putative polyphosphate kinase 1 predicted to be principally responsible for poly-P synthesis. We generated a targeted
ppk1
deletion mutant (Δ
ppk1
) in
C. jejuni
strain 81-176 and found that Δ
ppk1
, as well as the Δ
spoT
stringent response mutant, exhibited low levels of poly-P at all growth stages. In contrast, wild-type
C. jejuni
poly-P levels increased significantly as the bacteria transitioned from log to stationary phase. Phenotypic analyses revealed that the Δ
ppk1
mutant was defective for survival during osmotic shock and low-nutrient stress. However, certain phenotypes associated with
ppk1
deletion in other bacteria (i.e., motility and oxidative stress) were unaffected in the
C. jejuni
Δ
ppk1
mutant, which also displayed an unexpected increase in biofilm formation. The
C. jejuni
Δ
ppk1
mutant was also defective for the virulence-associated phenotype of intraepithelial cell survival in a tissue culture infection model and exhibited a striking, dose-dependent chick colonization defect. These results indicate that poly-P utilization and accumulation contribute significantly to
C. jejuni
pathogenesis and affect its ability to adapt to specific stresses and stringencies. Furthermore, our study demonstrates that poly-P likely plays both similar and unique roles in
C. jejuni
compared to its roles in other bacteria and that poly-P metabolism is linked to stringent response mechanisms in
C. jejuni
.
The Plant Pathogen
Pantoea ananatis
Produces
N
-Acylhomoserine Lactone and Causes Center Rot Disease of Onion by Quorum Sensing
Authors: Morohoshi Tomohiro , Nakamura Yuta , Yamazaki Go , Ishida Akio , Kato Norihiro , Ikeda Tsukasa
Published: September 2007

A number of gram-negative bacteria have a quorum-sensing system and produce
N
-acyl-
l
-homoserine lactone (AHL) that they use them as a quorum-sensing signal molecule.
Pantoea ananatis
is reported as a common colonist of wheat heads at ripening and causes center rot of onion. In this study, we demonstrated that
P. ananatis
SK-1 produced two AHLs,
N
-hexanoyl-
l
-homoserine lactone (C6-HSL) and
N
-(3-oxohexanoyl)-
l
-homoserine lactone (3-oxo-C6-HSL). We cloned the AHL-synthase gene (
eanI
) and AHL-receptor gene (
eanR
) and revealed that the deduced amino acid sequence of EanI/EanR showed high identity to those of EsaI/EsaR from
P. stewartii
. EanR repressed the
ean
box sequence and the addition of AHLs resulted in derepression of
ean
box. Inactivation of the chromosomal
eanI
gene in SK-1 caused disruption of exopolysaccharide (EPS) biosynthesis, biofilm formation, and infection of onion leaves, which were recovered by adding exogenous 3-oxo-C6-HSL. These results demonstrated that the quorum-sensing system involved the biosynthesis of EPS, biofilm formation, and infection of onion leaves in
P. ananatis
SK-1.
Interplay between the Temperate Phages PY54 and N15, Linear Plasmid Prophages with Covalently Closed Ends
Authors: Hammerl Jens A. , Klein Iris , Appel Bernd , Hertwig Stefan
Published: September 2007

The objective of this study was to determine whether the temperate
Yersinia enterocolitica
phage PY54 may interact with the related
Escherichia coli
phage N15 during both the lysogenic and the lytic cycle in the same cell. The PY54 and N15 prophages are linear plasmids which have been shown to be compatible and stably replicating in
E. coli
and
Yersinia
. In
E. coli
, the PY54 prophage does not restrict N15 propagation. In contrast, N15 reduces by use of its
cor
gene the susceptibility of
Yersinia
strains to PY54. Doubly lysogenic
E. coli
strains release PY54 virions, some of which apparently contain the N15 genome. Further experiments with replicative miniplasmid derivatives of PY54, N15, and the related
Klebsiella oxytoca
phage φKO2 demonstrated that the φKO2 and N15 plasmid prophages belong to the same incompatibility group.
The CbiB Protein of
Salmonella enterica
Is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway
Authors: Zayas Carmen L. , Claas Kathy , Escalante-Semerena Jorge C.
Published: September 2007

We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B
12
) pathway of
Salmonella enterica
serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but
l
-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.
The Alanine Racemase of
Mycobacterium smegmatis
Is Essential for Growth in the Absence of
d
-Alanine
Authors: Milligan Daniel L. , Tran Sieu L. , Strych Ulrich , Cook Gregory M. , Krause Kurt L.
Published: September 2007

Alanine racemase, encoded by the gene
alr
, is an important enzyme in the synthesis of
d
-alanine for peptidoglycan biosynthesis. Strains of
Mycobacterium smegmatis
with a deletion mutation of the
alr
gene were found to require
d
-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of
d
-alanine for cell wall biosynthesis in
M. smegmatis
and confirms alanine racemase as a viable target gene for antimycobacterial drug development.
trans
-Acting Factors and
cis
Elements Involved in Glucose Repression of Arabinan Degradation in
Bacillus subtilis
Authors: Inácio José Manuel , de Sá-Nogueira Isabel
Published: September 2007

In
Bacillus subtilis
, the synthesis of enzymes involved in the degradation of arabinose-containing polysaccharides is subject to carbon catabolite repression (CCR). Here we show that CcpA is the major regulator of repression of the arabinases genes in the presence of glucose. CcpA acts via binding to one
cre
each in the promoter regions of the
abnA
and
xsa
genes and to two
cre
s in the
araABDLMNPQ-abfA
operon. The contributions of the coeffectors HPr and Crh to CCR differ according to growth phase. HPr dependency occurs during both exponential growth and the transitional phase, while Crh dependency is detected mainly at the transitional phase. Our results suggest that Crh synthesis may increase at the end of exponential growth and consequently contribute to this effect, together with other factors.
Combined transmyocardial revascularization and cell-based angiogenic gene therapy increases transplanted cell survival
Authors: Spiegelstein Dan , Kim Christopher , Zhang Yaoguang , Li Guangming , Weisel Richard D. , Li Ren-Ke , Yau Terrence M.
Published: September 2007

We hypothesized that pretreatment of an infarcted heart by mechanical transmyocardial revascularization (TMR) before transplantation of bone marrow cells (BMCs) or BMC-expressing angiogenic growth factors would increase transplanted BMC survival and enhance myocardial repair. Female Lewis rats underwent coronary ligation 3 wk before creation of 10 needle TMR channels (3 groups) or no TMR (3 groups), followed by transplantation of 3 × 106male donor BMCs, BMC transfected with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1) (BMC + VBI), or medium alone. At 1, 3, and 7 days, we evaluated transplanted cell survival, vascular densities, and left ventricular (LV) function ( N = 4 per group × 6 groups × 3 time points). At 3 days, vascular densities in the scar were increased by TMR + BMC + VBI and by BMC + VBI ( P < 0.05), and at 7 days, vascular densities were greatest in rats receiving TMR + BMC + VBI ( P < 0.05). Transplanted cell survival at 3 and 7 days was increased by TMR and by BMC + VBI. Combined therapy with TMR + BMC + VBI resulted in the greatest cell survival at 3 days ( P < 0.05) versus BMC. After 7 days, LV ejection fraction (LVEF) was lowest in rats receiving neither BMC nor TMR and greatest in rats receiving TMR + BMC + VBI ( P = 0.004). We concluded that mechanical pretreatment of infarcted myocardium by TMR enhances the effect of subsequent cell-based gene therapy on transplanted cell survival, angiogenesis, and LV function. Scar pretreatment with TMR combined with cell-based multigene therapy may maximize myocardial repair.
Arterial remodeling in response to hypertension using a constituent-based model
Authors: Tsamis Alkiviadis , Stergiopulos Nikos
Published: September 2007

Hypertension-induced arterial remodeling has been previously modeled using stress-driven remodeling rate equations in terms of global geometrical adaptation (Rachev A, Stergiopulos N, Meister JJ. Theoretical study of dynamics of arterial wall remodeling in response to changes in blood pressure. J Biomech 29: 635–642, 1996) and was extended later to include adaptation of material properties (Rachev A, Stergiopulos N, Meister JJ. A model for geometric and mechanical adaptation of arteries to sustained hypertension. J Biomech Eng 120: 9–17, 1998). These models, however, used a phenomenological strain energy function (SEF), the parameters of which do not bear a clear physiological meaning. Here, we extend the work of Rachev et al. (1998) by applying similar remodeling rate equations to a constituent-based SEF. The new SEF includes a statistical description for collagen engagement, and remodeling now affects material properties only through changes in the collagen engagement probability density function. The model predicts asymptotic wall thickening and unchanged deformed inner radius as to conserve hoop stress and intimal shear stress, respectively, at the final adapted hypertensive state. Mechanical adaptation serves to restore arterial compliance to control levels. Average circumferential stress-strain curves show that the material at the final adapted hypertensive state is softer than its normotensive counterpart. These findings as well as the predicted pressure-diameter curves are in good qualitative agreement with experimental data. The novelty in our findings is that biomechanical adaptation leading to maintenance of compliance at the hypertensive state can be perfectly achieved by appropriate readjustment of the collagen engagement profile alone.
Mitochondrial arginase II constrains endothelial NOS-3 activity
Authors: Lim Hyun Kyo , Lim Hyun Kyoung , Ryoo Sungwoo , Benjo Alex , Shuleri Karl , Miriel Victor , Baraban Ezra , Camara Andre , Soucy Kevin , Nyhan Daniel , Shoukas Artin , Berkowitz Dan E.
Published: September 2007

Emerging evidence supports the idea that arginase, expressed in the vascular endothelial cells of humans and other species, modulates endothelial nitric oxide (NO) synthase-3 (NOS-3) activity by regulating intracellular l-arginine bioavailability. Arginase II is thought to be expressed in the mitochondria of a variety of nonendothelial cells, whereas arginase I is known to be confined to the cytosol of hepatic and other cells. The isoforms that regulate NOS-3 and their subcellular distribution, however, remain incompletely characterized. We therefore tested the hypothesis that arginase II is confined to the mitochondria and that mitochondrial arginase II reciprocally regulates vascular endothelial NO production. Western blot analysis, immunocytochemistry with MitoTracker, and immunoelectron microscopy confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly decreased, whereas NO production was significantly increased in the aorta and isolated endothelial cells from arginase II knockout (ArgII−/−) mice compared with wild-type (WT) mice. The vasorelaxation response to acetylcholine (ACh) was markedly enhanced and the vasoconstrictor response to phenylephrine (PE) attenuated in ArgII−/− in pressurized mouse carotid arteries. Furthermore, inhibition of NOS-3 by NG-nitro-l-arginine methyl ester (l-NAME) impaired ACh response and restored the PE response to that observed in WT vessels. Vascular stiffness, as assessed by pulse wave velocity (PWV), was significantly decreased in ArgII−/− compared with WT mice. On the other hand, 14 days of oral l-NAME treatment significantly increased PWV in both WT and ArgII−/− mice, such that they were not significantly different from one another. These data suggest that arginase II is predominantly confined to the mitochondria and that this mitochondrial arginase II regulates NO production, vascular endothelial function, and vascular stiffness by modulating NOS-3 activity.
Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition
Authors: Tang Jen-Ruey , Seedorf Gregory , Balasubramaniam Vivek , Maxey Anne , Markham Neil , Abman Steven H.
Published: September 2007

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% ( P < 0.005) and 21% ( P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age ( P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age ( P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.
Normal prion protein trafficking in cultured human erythroblasts
Authors: Griffiths Rebecca E. , Heesom Kate J. , Anstee David J.
Published: September 2007

Normal prion protein (PrPc), an essential substrate for development of prion disease, is widely distributed in hematopoietic cells. Recent evidence that variant Creutzfeldt-Jakob disease can be transmitted by transfusion of red cell preparations has highlighted the need for a greater understanding of the biology of PrPc in blood and blood-forming tissues. Here, we show that in contrast to another glycosylphosphoinositol-anchored protein CD59, PrPc at the cell surface of cultured human erythroblasts is rapidly internalized through the endosomal pathway, where it colocalizes with the tetraspanin CD63. In the plasma membrane, PrPc colocalizes with the tetraspanin CD81. Cross-linking with anti-PrPc or anti-CD81 causes clustering of PrPc and CD81, suggesting they can share the same microdomain. These data are consistent with a role for tetraspanin-enriched microdomains in trafficking of PrPc. These results, when taken together with recent evidence that exosomes released from cells as a result of endosomal-mediated recycling to the plasma membrane contain prion infectivity, provide a pathway for the propagation of prion diseases.
Preemptive HMG-CoA reductase inhibition provides graft-versus-host disease protection by Th-2 polarization while sparing graft-versus-leukemia activity
Authors: Zeiser Robert , Youssef Sawsan , Baker Jeanette , Kambham Neeraja , Steinman Lawrence , Negrin Robert S.
Published: September 2007

We investigated whether atorvastatin (AT) was capable of protecting animals from acute graft-versus-host disease (aGVHD) across major histocompatibility complex (MHC) mismatch barriers. AT treatment of the donor induced a Th-2 cytokine profile in the adoptively transferred T cells and reduced their in vivo expansion, which translated into significantly reduced aGVHD lethality. Host treatment down-regulated costimulatory molecules and MHC class II expression on recipient antigen-presenting cells (APCs) and enhanced the protective statin effect, without impacting graft-versus-leukemia (GVL) activity. The AT effect was partially reversed in STAT6−/− donors and abrogated by L-mevalonate, indicating the relevance of STAT6 signaling and the L-mevalonate pathway for AT-mediated aGVHD protection. AT reduced prenylation levels of GTPases, abolished T-bet expression, and increased c-MAF and GATA-3 protein in vivo. Thus, AT has significant protective impact on aGVHD lethality by Th-2 polarization and inhibition of an uncontrolled Th-1 response while maintaining GVL activity, which is of great clinical relevance given the modest toxicity profile of AT.
Protein subcellular localization prediction based on compartment-specific features and structure conservation
Authors: Su Emily Chia-Yu , Chiu Hua-Sheng , Lo Allan , Hwang Jenn-Kang , Sung Ting-Yi , Hsu Wen-Lian
Published: September 2007

Background

Protein subcellular localization is crucial for genome annotation, protein function prediction, and drug discovery. Determination of subcellular localization using experimental approaches is time-consuming; thus, computational approaches become highly desirable. Extensive studies of localization prediction have led to the development of several methods including composition-based and homology-based methods. However, their performance might be significantly degraded if homologous sequences are not detected. Moreover, methods that integrate various features could suffer from the problem of low coverage in high-throughput proteomic analyses due to the lack of information to characterize unknown proteins.

Results

We propose a hybrid prediction method for Gram-negative bacteria that combines a one-versus-one support vector machines (SVM) model and a structural homology approach. The SVM model comprises a number of binary classifiers, in which biological features derived from Gram-negative bacteria translocation pathways are incorporated. In the structural homology approach, we employ secondary structure alignment for structural similarity comparison and assign the known localization of the top-ranked protein as the predicted localization of a query protein. The hybrid method achieves overall accuracy of 93.7% and 93.2% using ten-fold cross-validation on the benchmark data sets. In the assessment of the evaluation data sets, our method also attains accurate prediction accuracy of 84.0%, especially when testing on sequences with a low level of homology to the training data. A three-way data split procedure is also incorporated to prevent overestimation of the predictive performance. In addition, we show that the prediction accuracy should be approximately 85% for non-redundant data sets of sequence identity less than 30%.

Conclusion

Our results demonstrate that biological features derived from Gram-negative bacteria translocation pathways yield a significant improvement. The biological features are interpretable and can be applied in advanced analyses and experimental designs. Moreover, the overall accuracy of combining the structural homology approach is further improved, which suggests that structural conservation could be a useful indicator for inferring localization in addition to sequence homology. The proposed method can be used in large-scale analyses of proteomes.