bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of recombinant bovine tumor necrosis factor‐α on hormone release in lactating cows
Authors: KUSHIBIKI Shiro , SHINGU Hiroyuki , KOMATSU Tokushi , ITOH Fumiaki , KASUYA Etsuko , ASO Hisashi , HODATE Koichi
Published: December 2006

Tumor necrosis factor (TNF)‐α is a powerful macrophage cytokine released during infection, circulating in the blood to produce diverse effects in the organism. We examined the effect of recombinant bovine TNF‐α (rbTNF‐α) administration on hormone release in dairy cows during early lactation. Twelve non‐pregnant Holstein cows were treated subcutaneously with rbTNF‐α (2.5 µg/kg) or saline twice (at 11.00 and 23.00 hours). At 11.00 hours the next day, the cows were given growth hormone‐releasing hormone (GHRH, 0.25 µg/kg), thyrotrophin‐releasing hormone (TRH, 1.0 µg/kg), thyroid‐stimulating hormone (TSH, 10 µg/kg) or adrenocorticotropic hormone (500 µg/head) via the jugular vein. In the growth hormone‐releasing hormone challenge, the plasma growth hormone concentration was lower in the rbTNF‐α group than in the control (saline) group. The growth hormone and TSH responses to TRH were also smaller in the rbTNF‐α group than in the control. The plasma prolactin response to TRH was not affected by the rbTNF‐α treatment. In the TSH challenge, the rbTNF‐α‐treated cows had lower responses, as measured by plasma triiodothyronine and thyroxine, than the control cows. The rbTNF‐α treatment produced an increase in the basal plasma cortisol level, but the cortisol response to adrenocorticotropic hormone was the same level in both groups. The plasma concentrations of TNF‐α and interleukin‐1β in the cows were elevated by the rbTNF‐α treatment. The milk yield was reduced by the rbTNF‐α administration during 4 days. These data demonstrate that TNF‐α alters the secretion of pituitary and thyroid hormones in lactating cows. This effect may contribute to the suppression of the lactogenic function of the mammary gland observed in cases of coliform mastitis with high circulating TNF‐α levels.
Cre-
lox
-Based System for Multiple Gene Deletions and Selectable-Marker Removal in
Lactobacillus plantarum
Authors: Lambert Jolanda M. , Bongers Roger S. , Kleerebezem Michiel
Published: December 2006

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-
lox
have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-
lox
-based system for multiple gene deletions in
Lactobacillus plantarum
that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a
lox66
-P
32
-
cat-lox71
cassette, where
lox66
and
lox71
are mutant variants of
loxP
and P
32
-
cat
is a chloramphenicol resistance cassette. The
lox
sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the
lox66
-P
32
-
cat-lox71
cassette into a double-mutant
loxP
site, called
lox72
, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-
lox
-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the
melA
and
bsh1
loci on the chromosome of the gram-positive model organism
Lactobacillus plantarum
WCFS1. Furthermore, the efficiency of the Cre-
lox
-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci
melA
and
lacS2
in
L. plantarum
WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-
lox
resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.
Physiological, Ecological, and Phylogenetic Characterization of
Stappia
, a Marine CO-Oxidizing Bacterial Genus
Authors: Weber Carolyn F. , King Gary M.
Published: December 2006

Bacteria play a major role in marine CO cycling, yet very little is known about the microbes involved. Thirteen CO-oxidizing
Stappia
isolates obtained from existing cultures, macroalgae, or surf samples representing geographically and ecologically diverse habitats were characterized using biochemical, physiological, and phylogenetic approaches. All isolates were aerobic chemoorganotrophs that oxidized CO at elevated (1,000 ppm) and ambient-to-subambient concentrations (<0.3 ppm). All contained the form I (OMP)
coxL
gene for aerobic CO dehydrogenase and also the form II (BMS) putative
coxL
gene. In addition, some strains possessed
cbbL
, the large subunit gene for ribulose-1,5-bisphosphate carboxylase/oxygenase, suggesting the possibility of lithotrophic or mixotrophic metabolism. All isolates used a wide range of sugars, organic acids, amino acids, and aromatics for growth and grew at salinities from 5 to 45 ppt. All but one isolate denitrified or respired nitrate. Phylogenetic analyses based on 16S rRNA gene sequences indicated that several isolates could not be distinguished from
Stappia aggregata
and contributed to a widely distributed species complex. Four isolates (of strains GA15, HI, MIO, and M4) were phylogenetically distinct from validly described
Stappia
species and closely related genera (e.g.,
Ahrensia
,
Pannonibacter
,
Pseudovibrio
, and
Roseibium
). Substrate utilization profiles, enzymatic activity, and membrane lipid composition further distinguished these isolates and supported their designations as new
Stappia
species. The observed metabolic versatility of
Stappia
likely accounts for its cosmopolitan distribution and its ability to contribute to CO cycling as well as other important biogeochemical cycles.
Induction by Hypoxia of Heterologous-Protein Production with the Kl
PDC1
Promoter in Yeasts
Authors: Camattari Andrea , Bianchi Michele M. , Branduardi Paola , Porro Danilo , Brambilla Luca
Published: December 2006

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the Kl
PDC1
promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the Kl
PDC1
promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in
Kluyveromyces lactis
under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The
l
ac
Z
gene from
Escherichia coli
was cloned downstream of the Kl
PDC1
promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in
Saccharomyces cerevisiae
and in
Zygosaccharomyces bailii
, and effects of hypoxic induction were measured and compared to those already observed in
K. lactis
cells. Interestingly, we found that the induction still worked in
Z. bailii
; thus, this promotor constitutes a possible inducible system for this new nonconventional host.
The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of
Escherichia coli
Authors: Sletta H. , Tøndervik A. , Hakvåg S. , Aune T. E. Vee , Nedal A. , Aune R. , Evensen G. , Valla S. , Ellingsen T. E. , Brautaset T.
Published: December 2006

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-α2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of
Escherichia coli
. All three proteins were poorly expressed when put under control of the strong
Pm/xylS
promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence,
pelB
,
ompA
, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-α2b expression remained poor even when fused to a signal sequence, and an alternative IFN-α2b coding sequence that was optimized for effective expression in
Escherichia coli
was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in
E. coli
under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.
Role of Cytochrome
bd
Oxidase from
Corynebacterium glutamicum
in Growth and Lysine Production
Authors: Kabus Armin , Niebisch Axel , Bott Michael
Published: December 2006

Corynebacterium glutamicum
possesses two terminal oxidases, cytochrome
aa
3
and cytochrome
bd
. Cytochrome
aa
3
forms a supercomplex with the cytochrome
bc
1
complex, which contains an unusual diheme cytochrome
c
1
. Both the
bc
1
-
aa
3
supercomplex and cytochrome
bd
transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome
bd
for growth and lysine production. When cultivated in glucose minimal medium, a
cydAB
deletion mutant of
C. glutamicum
ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome
bd
oxidase in ATCC 13032 led to a reduction of the growth rate by ∼45% and of the maximal biomass by ∼35%, presumably as a consequence of increased electron flow through the inefficient cytochrome
bd
oxidase. In the
l
-lysine-producing
C. glutamicum
strain MH20-22B, deletion of the
cydAB
genes had only minor effects on growth rate and biomass formation, but lysine production was increased by ∼12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by
C. glutamicum
.
Induction of a Global Stress Response during the First Step of
Escherichia coli
Plate Growth
Authors: Cuny Caroline , Lesbats Maïalène , Dukan Sam
Published: December 2006

We have investigated the first events that occur when exponentially grown cells are transferred from a liquid medium (Luria-Bertani [LB]) to a solid medium (LB agar [LBA]). We observed an initial lag phase of 180 min for the wild type MG1655 without any apparent growth. This lack of growth was independent of the bacterial physiological state (either the stationary or the exponential phase), the solid medium composition, or the number of cells on the plate, but it was dependent on the bacterial genotype. Using
lacZ
-reporter fusions and two-dimensional electrophoresis analysis, we observed that when cells from exponential-phase cultures were plated on LBA, several global regulons, like heat shock regulons (RpoH, RpoE, CpxAR) and oxidative-stress regulons (SoxRS, OxyR, Fur), were immediately induced. Our results indicate that in order to grow on plates, bacteria must not only adapt to new conditions but also perceive a real stress.
Coupling ofMethanothermobacter thermautotrophicusMethane Formation and Growth in Fed-Batch and Continuous Cultures under Different H2Gassing Regimens
Authors: de Poorter Linda M. I. , Geerts Wim J. , Keltjens Jan T.
Published: December 2006

In nature, H2- and CO2-utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth,Methanothermobacter thermautotrophicuswas cultured in a fed-batch fermentor and in a chemostat under different 80% H2-20% CO2gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (pH2). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (YCH4max), notably approximately 3 and 7 g (dry weight) of methane formed mol−1, for growth under low (pH2< 12 kPa)- and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the “specific maintenance” requirements or, more likely, the degree of proton leakage and proton slippage processes. At lowpH2values, the “specific maintenance” diminished and the specific growth yields approachedYCH4max, indicating that growth and methanogenesis became fully coupled.
Characterization of a Large, Stable, High-Copy-Number
Streptomyces
Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction
Authors: Fong Ryan , Hu Zhihao , Hutchinson C. Richard , Huang Jianqiang , Cohen Stanley , Kao Camilla
Published: December 2006

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous
Streptomyces
host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene,
spd
, and a partition gene,
parAB
, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (
actII-ORF4
) and biosynthetic genes (
eryA
) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.
Recovery of
Mycobacterium bovis
from Soft Fresh Cheese Originating in Mexico
Authors: Harris N. Beth , Payeur Janet , Bravo Doris , Osorio Ruben , Stuber Tod , Farrell David , Paulson Debra , Treviso Scarlett , Mikolon Andrea , Rodriguez-Lainz Alfonso , Cernek-Hoskins Shannon , Rast Robert , Ginsberg Michele , Kinde Hailu
Published: December 2006

Recent outbreaks of human tuberculosis in the United States caused by
Mycobacterium bovis
have implicated cheese originating in Mexico as a source of these infections. A total of 203 samples of cheese originating in Mexico were cultured, and
M. bovis
was recovered from one specimen. Therefore,
M. bovis
can be recovered from cheese and may be a source of human infections.
Differentiation of
Listeria monocytogenes
Serovars by Using Artificial Neural Network Analysis of Fourier-Transformed Infrared Spectra
Authors: Rebuffo-Scheer Cecilia A. , Schmitt Jürgen , Scherer Siegfried
Published: December 2006

A classification system based on Fourier transform infrared (FTIR) spectroscopy combined with artificial neural network analysis was designed to differentiate 12 serovars of
Listeria monocytogenes
using a reference database of 106 well-defined strains. External validation was performed using a test set of another 166
L. monocytogenes
strains. The O antigens (serogroup) of 164 strains (98.8%) could be identified correctly, and H antigens were correctly determined in 152 (91.6%) of the test strains. Importantly, 40 out of 41 potentially epidemic serovar 4b strains were unambiguously identified. FTIR analysis is superior to PCR-based systems for serovar differentiation and has potential for the rapid, simultaneous identification of both species and serovar of an unknown
Listeria
isolate by simply measuring a whole-cell infrared spectrum.
Isolation of
Rickettsia rhipicephali
and
Rickettsia bellii
from
Haemaphysalis juxtakochi
Ticks in the State of São Paulo, Brazil
Authors: Labruna Marcelo B. , Pacheco Richard C. , Richtzenhain Leonardo J. , Szabó Matias P. J.
Published: December 2006

In the present study, attempts to isolate
Rickettsia
in cell culture were performed individually in seven specimens of
Haemaphysalis juxtakochi
ticks collected in the state of São Paulo (southeastern Brazil).
Rickettsia
was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific
Rickettsia
primers targeting the genes
gltA
,
htrA
,
ompA
, and
ompB
. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as
Rickettsia bellii
(isolates HJ#1, HJ#2, HJ#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to
Rickettsia
sp. strain R300, previously detected in
H. juxtakochi
in northern Brazil, and to
Rickettsia rhipicephali
, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of
Rickettsia
isolates, both isolate HJ#5 and strain R300 were identified as South American strains of
R. rhipicephali
, which was confirmed in this continent for the first time. Isolation of
R. bellii
from
H. juxtakochi
ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that
R. bellii
is indeed the most frequent
Rickettsia
species infecting ticks in Brazil. Currently, the role of both
R. rhipicephali
and
R. bellii
as human pathogens is regarded as unknown.
A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides inGeobacillus stearothermophilus
Authors: Shulami Smadar , Zaide Galia , Zolotnitsky Gennady , Langut Yael , Feld Geoff , Sonenshein Abraham L. , Shoham Yuval
Published: December 2006

Geobacillus stearothermophilusT-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). ThexynDpromoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His6-XynC, bound to a 220-bp fragment corresponding to thexynEoperator. DNase I footprinting analysis showed four protected zones that cover the −53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated thatxynEtranscription is activated 140-fold in the presence of 1.5 μM XynC. The His6-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (ΔCp) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system.
Novel Quantitative Biosystem for Modeling Physiological Fluid Shear Stress on Cells
Authors: Nauman Eric A. , Ott C. Mark , Sander Ed , Tucker Don L. , Pierson Duane , Wilson James W. , Nickerson Cheryl A.
Published: December 2006

The response of microbes to changes in the mechanical force of fluid shear has important implications for pathogens, which experience wide fluctuations in fluid shear in vivo during infection. However, the majority of studies have not cultured microbes under physiological fluid shear conditions within a range commonly encountered by microbes during host-pathogen interactions. Here we describe a convenient batch culture biosystem in which (i) the levels of fluid shear force can be varied within physiologically relevant ranges and quantified via mathematical models and (ii) large numbers of cells can be planktonically grown and harvested to examine the effect of fluid shear levels on microbial genomic and phenotypic responses. A quantitative model based on numerical simulations and in situ imaging analysis was developed to calculate the fluid shear imparted by spherical beads of different sizes on bacterial cell cultures grown in a rotating wall vessel (RWV) bioreactor. To demonstrate the application of this model, we subjected cultures of the bacterial pathogen
Salmonella enterica
serovar Typhimurium to three physiologically-relevant fluid shear ranges during growth in the RVW and demonstrated a progressive relationship between the applied fluid shear and the bacterial genetic and phenotypic responses. By applying this model to different cell types, including other bacterial pathogens, entire classes of genes and proteins involved in cellular interactions may be discovered that have not previously been identified during growth under conventional culture conditions, leading to new targets for vaccine and therapeutic development.
Role of Protein
O
-Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of
Cryptococcus neoformans
Authors: Olson Gillian M. , Fox Deborah S. , Wang Ping , Alspaugh J. Andrew , Buchanan Kent L.
Published: December 2006

Protein O mannosylation is initiated in the endoplasmic reticulum by protein
O
-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen
Cryptococcus neoformans
. Disruption of the
C. neoformans PMT4
gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the
pmt4
mutant compared to wild-type cells. The
pmt4
mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the
pmt4
mutant, as well as a global decrease in protein mannosylation. Heterologous expression of
C. neoformans PMT4
in a
Saccharomyces cerevisiae pmt1pmt4
mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in
C. neoformans
was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of
C. neoformans
.
Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists:
Leishmania
-Specific Informative Sites in Hydrophilic Loops of Trans- Endoplasmic Reticulum
N
-Acetylglucosamine-1-Phosphate Transferase
Authors: Waki Kayoko , Dutta Sujoy , Ray Debalina , Kolli Bala Krishna , Akman Leyla , Kawazu Shin-Ichiro , Lin Chung-Ping , Chang Kwang-Poo
Published: December 2006

A sequence database was created for the
Leishmania N
-acetylglucosamine-1-phosphate transferase (
nagt
) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven
nagt
sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26
nagt
sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus
Leishmania
(
L. amazonensis-L. mexicana
,
L. donovani-L. infantum
,
L. tropica
,
L. major
, and
L. turanica-L. gerbilli
), subgenus
Viannia
(
L. braziliensis
,
L. panamensis
), and one unclassified (
L. enriettii
) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of
nagt
varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to
Leishmania
evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in
L. tropica
but not in
L. donovani-L. infantum.
This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of
nagt
are restricted largely to several extended hydrophilic loops predicted from hypothetical models of
Leishmania
NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of
nagt
from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.
Expression of
Magnaporthe grisea
Avirulence Gene
ACE1
Is Connected to the Initiation of Appressorium-Mediated Penetration
Authors: Fudal Isabelle , Collemare Jérôme , Böhnert Heidi U. , Melayah Delphine , Lebrun Marc-Henri
Published: December 2006

Magnaporthe grisea
is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize
M. grisea
signals corresponding to specific secreted proteins encoded by avirulence genes. The
M. grisea ACE1
avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the
Pi33
resistance gene. Using a transcriptional fusion between
ACE1
promoter and
eGFP
, we showed that
ACE1
is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes.
ACE1
is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes.
ACE1
expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants Δ
cpkA
and Δ
mac1 sum1
-
99
and tetraspanin mutant Δ
pls1
::
hph
differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves.
ACE1
is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event.
ACE1
is not expressed in appressoria of the
buf1
::
hph
mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to
buf1
::
hph
appressoria restores appressorial development and
ACE1
expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and
ACE1
expression. These experiments suggest that
ACE1
appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.
Rapid Evolution of Diminished Transformability in
Acinetobacter baylyi
Authors: Bacher Jamie M. , Metzgar David , de Crécy-Lagard Valérie
Published: December 2006

The reason for genetic exchange remains a crucial question in evolutionary biology.
Acinetobacter baylyi
strain ADP1 is a highly competent and recombinogenic bacterium. We compared the parallel evolution of wild-type and engineered noncompetent lineages of
A. baylyi
in the laboratory. If transformability were to result in an evolutionary benefit, it was expected that competent lineages would adapt more rapidly than noncompetent lineages. Instead, regardless of competency, lineages adapted to the same extent under several laboratory conditions. Furthermore, competent lineages repeatedly evolved a much lower level of transformability. The loss of competency may be due to a selective advantage or the irreversible transfer of loss-of-function alleles of genes required for transformation within the competent population.
Proteolytic Processing Is Not Essential for Multiple Functions of the
Escherichia coli
Autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I)
Authors: Charbonneau Marie-Ève , Berthiaume Frédéric , Mourez Michael
Published: December 2006

The
Escherichia coli
adhesin involved in diffuse adherence (AIDA-I), like many other autotransporter proteins, is released in the periplasm as a proprotein undergoing proteolytic processing after its translocation across the outer membrane. The proprotein is cleaved into a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with the outer membrane and can be released by heat treatment. The mechanism of cleavage of the proprotein and its role in the functionality of AIDA-I are not understood. Here, we show that cleavage is independent of the amount of AIDA-I in the outer membrane, suggesting an intramolecular autoproteolytic mechanism or a cleavage mediated by an unknown protease. We show that the two fragments, mature AIDA-I and AIDAc, can be cosolubilized and copurified in a folded and active conformation. We observed that the release by heat treatment results from the unfolding of AIDA-I and that the interaction of AIDA-I with AIDAc seems to be disturbed only by denaturation. We constructed an uncleavable point mutant of AIDA-I, where a serine of the cleavage site was changed into a leucine, and showed that adhesion, autoaggregation, and biofilm formation mediated by the mutant are indistinguishable from the wild-type levels. Lastly, we show that both proteins can mediate the invasion of cultured epithelial cells. Taken together, our experiments suggest that the proteolytic processing of AIDA-I plays a minor role in the functionality of this protein.
SarA Is a Repressor of
hla
(α-Hemolysin) Transcription in
Staphylococcus aureus
: Its Apparent Role as an Activator of
hla
in the Prototype Strain NCTC 8325 Depends on Reduced Expression of
sarS
Authors: Oscarsson Jan , Kanth Anna , Tegmark-Wisell Karin , Arvidson Staffan
Published: December 2006

In most
Staphylococcus aureus
strains, inactivation of
sarA
increases
hla
transcription, indicating that
sarA
is a repressor. However, in
S. aureus
NCTC 8325 and its derivatives, used for most studies of
hla
regulation, inactivation of
sarA
resulted in decreased
hla
transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its
rsbU
mutation, which leads to σ
B
deficiency. This has now been verified by the demonstration that
sarA
repressed
hla
transcription in an
rsbU
+
derivative of strain 8325-4 (SH1000). That
sarA
could act as a repressor of
hla
in an 8325-4 background was confirmed by the observation that inactivation of
sarA
in an
agr sarS rot
triple mutant dramatically increased
hla
transcription to wild-type levels. However, the apparent role of
sarA
as an activator of
hla
in 8325-4 was not a result of the
rsbU
mutation alone, as inactivation of
sarA
in another
rsbU
mutant, strain V8, led to increased
hla
transcription. Northern blot analysis revealed much higher levels of
sarS
mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the
sarS
activator,
tcaR
, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in
sarS
transcription upon the inactivation of
sarA
was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of
sarA
in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of
sarA
means a net decrease in repressor activity and, therefore, enhanced
hla
transcription.
Release of Immunity Protein Requires Functional Endonuclease Colicin Import Machinery
Authors: Duché Denis , Frenkian Aurélie , Prima Valérie , Lloubès Roland
Published: December 2006

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.
Mycoplasma genitalium
P140 and P110 Cytadhesins Are Reciprocally Stabilized and Required for Cell Adhesion and Terminal-Organelle Development
Authors: Burgos Raul , Pich Oscar Q. , Ferrer-Navarro Mario , Baseman Joel B. , Querol Enrique , Piñol Jaume
Published: December 2006

Mycoplasma genitalium
is a human pathogen that mediates cell adhesion by a complex structure known as the attachment organelle. This structure is composed of cytadhesins and cytadherence-associated proteins, but few data are available about the specific role of these proteins in
M. genitalium
cytadherence. We have deleted by homologous recombination the mg191 and mg192 genes from the MgPa operon encoding the P140 and P110 cytadhesins. Molecular characterization of these mutants has revealed a reciprocal posttranslational stabilization between the two proteins. Loss of either P140 or P110 yields a hemadsorption-negative phenotype and correlates with decreased or increased levels of cytoskeleton-related proteins MG386 and DnaK, respectively. Scanning electron microscopy analysis reveals the absolute requirement of P140 and P110 for the proper development of the attachment organelle. The phenotype described for these mutants resembles that of the spontaneous class I and class II cytadherence-negative mutants [G. R. Mernaugh, S. F. Dallo, S. C. Holt, and J. B. Baseman, Clin. Infect. Dis.
17
(Suppl. 1)
:
S69-S78, 1993], whose genetic basis remained undetermined until now. Complementation assays and sequencing analysis demonstrate that class I and class II mutants are the consequence of large deletions affecting the mg192 and mg191-mg192 genes, respectively. These deletions originated from single-recombination events involving sequences of the MgPa operon and the MgPa island located immediately downstream. We also demonstrate the translocation of MgPa sequences to a particular MgPa island by double-crossover events. Based on these observations, we propose that in addition to being a source of antigenic variation, MgPa islands could be also involved in a general phase variation mechanism switching on and off, in a reversible or irreversible way, the adhesion properties of
M. genitalium
.
The OxyR Regulon in Nontypeable
Haemophilus influenzae
Authors: Harrison Alistair , Ray William C. , Baker Beth D. , Armbruster David W. , Bakaletz Lauren O. , Munson Robert S.
Published: December 2006

Nontypeable
Haemophilus influenzae
(NTHi) is a gram-negative bacterium and a common commensal organism of the upper respiratory tract in humans. NTHi causes a number of diseases, including otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During the course of colonization and infection, NTHi must withstand oxidative stress generated by insult due to multiple reactive oxygen species produced endogenously by other copathogens and by host cells. Using an NTHi-specific microarray containing oligonucleotides representing the 1821 open reading frames of the recently sequenced NTHi isolate 86-028NP, we have identified 40 genes in strain 86-028NP that are upregulated after induction of oxidative stress due to hydrogen peroxide. Further comparisons between the parent and an isogenic
oxyR
mutant identified a subset of 11 genes that were transcriptionally regulated by OxyR, a global regulator of oxidative stress. Interestingly, hydrogen peroxide induced the OxyR-independent upregulation of expression of the genes encoding components of multiple iron utilization systems. This finding suggested that careful balancing of levels of intracellular iron was important for minimizing the effects of oxidative stress during NTHi colonization and infection and that there are additional regulatory pathways involved in iron utilization.
Lysine Decarboxylase Expression by
Vibrio vulnificus
Is Induced by SoxR in Response to Superoxide Stress
Authors: Kim Ju-Sim , Choi Sang Ho , Lee Jeong K.
Published: December 2006

Lysine decarboxylase expression by
Vibrio vulnificus
, which is up-regulated by CadC in response to acid stress, is also induced by SoxR in response to superoxide stress. SoxR binds to the promoter region of the
cadBA
operon, coding for a lysine-cadaverine antiporter (CadB) and a lysine decarboxylase (CadA). The induction of
cadBA
transcription by SoxR is independent of CadC. Cadaverine, which neutralizes the external medium, also appears to scavenge superoxide radicals, since increasing cellular cadaverine by elevating the gene dosage of
cadBA
significantly diminished the induction of Mn-containing superoxide dismutase under methyl viologen-induced oxidative stress. Consistently, a lack of cadaverine caused by mutation in
cadA
resulted in low tolerance to oxidative stress compared with that of the wild type.
Proteomic Analysis and Identification of
Streptococcus pyogenes
Surface-Associated Proteins
Authors: Severin Anatoly , Nickbarg Elliott , Wooters Joseph , Quazi Shakey A. , Matsuka Yury V. , Murphy Ellen , Moutsatsos Ioannis K. , Zagursky Robert J. , Olmsted Stephen B.
Published: December 2006

Streptococcus pyogenes
is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of
S. pyogenes
. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in
S. pyogenes
. Several proteins were expressed in
Escherichia coli
, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzyme-linked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by
S. pyogenes
.
Overlapping and Specialized Functions of the Molybdenum-Dependent Regulators MopA and MopB in
Rhodobacter capsulatus
Authors: Wiethaus Jessica , Wirsing Andrea , Narberhaus Franz , Masepohl Bernd
Published: December 2006

The phototrophic purple bacterium
Rhodobacter capsulatus
encodes two similar but functionally not identical molybdenum-dependent regulator proteins (MopA and MopB), which are known to replace each other in repression of the
modABC
genes (coding for an ABC-type high-affinity Mo transport system) and
anfA
(coding for the transcriptional activator of Fe-nitrogenase genes). We identified further Mo-regulated (
mor
) genes coding for a putative ABC-type transport system of unknown function (MorABC) and a putative Mo-binding protein (Mop). The genes coding for MopA and the ModABC transporter form part of a single transcriptional unit,
mopA
-
modABCD
, as shown by reverse transcriptase PCR. Immediately upstream of
mopA
and transcribed in the opposite direction is
mopB
. The genes coding for the putative MorABC transporter belong to two divergently transcribed operons,
morAB
and
morC
. Expression studies based on
lacZ
reporter gene fusions in mutant strains defective for either MopA, MopB, or both revealed that the regulators substitute for each other in Mo-dependent repression of
morAB
and
morC
. Specific Mo-dependent activation of the
mop
gene by MopA, but not MopB, was found to control the putative Mo-binding protein. Both MopA and MopB are thought to bind to conserved DNA sequences with dyad symmetry in the promoter regions of all target genes. The positions of these so-called Mo boxes relative to the transcription start sites (as determined by primer extension analyses) differed between Mo-repressed genes and the Mo-activated
mop
gene. DNA mobility shift assays showed that MopA and MopB require molybdenum to bind to their target sites with high affinity.
A Conserved Histidine in Cytochrome
c
Maturation Permease CcmB of
Shewanella putrefaciens
Is Required for Anaerobic Growth below a Threshold Standard Redox Potential
Authors: Dale Jason R. , Wade Roy , DiChristina Thomas J.
Published: December 2006

Shewanella putrefaciens
strain 200 respires a wide range of compounds as terminal electron acceptor. The respiratory versatility of
Shewanella
is attributed in part to a set of
c
-type cytochromes with widely varying midpoint redox potentials (E′
0
). A point mutant of
S. putrefaciens
, originally designated Urr14 and here renamed CCMB1, was found to grow at wild-type rates on electron acceptors with high E′
0
[O
2
, NO
3
−
, Fe(III) citrate, MnO
2
, and Mn(III) pyrophosphate] yet was severely impaired for growth on electron acceptors with low E′
0
[NO
2
−
, U(VI), dimethyl sulfoxide, TMAO (trimethylamine
N
-oxide), fumarate, γ-FeOOH, SO
3
2−
, and S
2
O
3
2−
]. Genetic complementation and nucleotide sequence analyses indicated that the CCMB1 respiratory mutant phenotype was due to mutation of a conserved histidine residue (H108Y) in a protein that displayed high homology to
Escherichia coli
CcmB, the permease subunit of an ABC transporter involved in cytochrome
c
maturation. Although CCMB1 retained the ability to grow on electron acceptors with high E′
0
, the cytochrome content of CCMB1 was <10% of that of the wild-type strain. Periplasmic extracts of CCMB1 contained slightly greater concentrations of the thiol functional group (-SH) than did the wild-type strain, an indication that the E
h
of the CCMB1 periplasm was abnormally low. A
ccmB
deletion mutant was unable to respire anaerobically on any electron acceptor, yet retained aerobic respiratory capability. These results suggest that the mutation of a conserved histidine residue (H108) in CCMB1 alters the redox homeostasis of the periplasm during anaerobic growth on electron acceptors with low (but not high) E′
0
. This is the first report of the effects of Ccm deficiencies on bacterial respiration of electron acceptors whose E′
0
nearly span the entire redox continuum.
Regulation of
Aggregatibacter
(
Actinobacillus
)
actinomycetemcomitans
Leukotoxin Secretion by Iron
Authors: Balashova Nataliya V. , Diaz Roger , Balashov Sergey V. , Crosby Juan A. , Kachlany Scott C.
Published: December 2006

The gram-negative oral and systemic pathogen
Aggregatibacter
(
Actinobacillus
)
actinomycetemcomitans
produces a leukotoxin (LtxA) that is a member of the RTX (repeats in toxin) family of secreted bacterial toxins. We have recently shown that LtxA has the ability to lyse erythrocytes, which results in a beta-hemolytic phenotype on Columbia blood agar. To determine if LtxA is regulated by iron, we examined beta-hemolysis under iron-rich and iron-limiting conditions. Beta-hemolysis was suppressed in the presence of FeCl
3
. In contrast, strong beta-hemolysis occurred in the presence of the iron chelator deferoxamine. We found that secretion of LtxA was completely inhibited by free iron, but expression of
ltxA
was not regulated by iron. Free chromium, cobalt, and magnesium did not affect LtxA secretion. Other LtxA-associated genes were not regulated by iron. Thus, iron appears to play an important role in the regulation of LtxA secretion in
A. actinomycetemcomitans
in a manner independent of gene regulation.