bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct and Indirect Effects of Protist Predation on Population Size Structure of a Bacterial Strain with High Phenotypic Plasticity
Authors: Corno Gianluca , Jürgens Klaus
Published: January 2006

We studied the impact of grazing and substrate supply on the size structure of a freshwater bacterial strain (Flectobacillussp.) which showed pronounced morphological plasticity. The cell length varied from 2 to >40 μm and encompassed rods, curved cells, and long filaments. Without grazers and with a sufficient substrate supply, bacteria grew mainly in the form of medium-sized rods (4 to 7 μm), with a smaller proportion (<10%) of filamentous forms. Grazing experiments with the bacterivorous flagellateOchromonassp. showed that freely suspended cells of <7 μm were highly vulnerable to grazers, whereas filamentous cells were resistant to grazing and became enriched during predation. A comparison of long-term growth in carbon-limited chemostats with and without grazers revealed that strikingly different bacterial populations developed: treatments with flagellates were composed of >80% filamentous cells. These attained a biomass comparable to that of populations in chemostats without grazers, which were composed of medium-sized rods and c-shaped cells. Carbon starvation resulted in a fast decrease in cell length and a shift towards small rods, which were highly vulnerable to grazing. Dialysis bag experiments in combination with continuous cultivation revealed that filament formation was significantly enhanced even without direct contact of bacteria with bacterivores and was thus probably stimulated by grazer excretory products.
Modulation of Rat Cecal Microbiota by Administration of Raffinose and Encapsulated
Bifidobacterium breve
Authors: Dinoto Achmad , Suksomcheep Akarat , Ishizuka Satoshi , Kimura Hanae , Hanada Satoshi , Kamagata Yoichi , Asano Kozo , Tomita Fusao , Yokota Atsushi
Published: January 2006

To investigate the effects of administration of raffinose and encapsulated
Bifidobacterium breve
JCM 1192
T
cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated
B. breve
(group CB), and basal diet supplemented with both raffinose and encapsulated
B. breve
(group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP).
B. breve
cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis.
B. breve
was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no
B. breve
signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of
Bifidobacterium animalis
, a
Bifidobacterium
indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.
Abundant and Diverse Fungal Microbiota in the Murine Intestine
Authors: Scupham Alexandra J , Presley Laura L. , Wei Bo , Bent Elizabeth , Griffith Natasha , McPherson Michael , Zhu Feilin , Oluwadara Oluwadayo , Rao Nagesh , Braun Jonathan , Borneman James
Published: January 2006

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla:Ascomycota,Basidiomycota,Chytridiomycota, andZygomycota. The largest assemblages of fungal rRNA sequences were related to the generaAcremonium,Monilinia,Fusarium,Cryptococcus/Filobasidium,Scleroderma,Catenomyces,Spizellomyces,Neocallimastix,Powellomyces,Entophlyctis,Mortierella, andSmittiumand the orderMucorales. The majority of bacterial rRNA gene clones were affiliated with the taxaBacteroidetes,Firmicutes,Acinetobacter, andLactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.
New
rfp
- and pES213-Derived Tools for Analyzing Symbiotic
Vibrio fischeri
Reveal Patterns of Infection and
lux
Expression In Situ
Authors: Dunn Anne K. , Millikan Deborah S. , Adin Dawn M. , Bose Jeffrey L. , Stabb Eric V.
Published: January 2006

Genetically altered or tagged
Vibrio fischeri
strains can be observed in association with their mutualistic host
Euprymna scolopes
, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in
V. fischeri
contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of
V. fischeri
plasmid pES213 were retained by ∼99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in
V. fischeri
. RFP expression did not detectably affect the fitness of
V. fischeri
, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most
E. scolopes
hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the
luxICDABEG
promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the
V. fischeri
-
E. scolopes
symbiosis.
The
Thermotoga maritima
Phenotype Is Impacted by Syntrophic Interaction with
Methanococcus jannaschii
in Hyperthermophilic Coculture
Authors: Johnson M. R. , Conners S. B. , Montero C. I. , Chou C. J. , Shockley K. R. , Kelly R. M.
Published: January 2006

Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium
Thermotoga maritima
when it was cocultured with the hyperthermophilic archaeon
Methanococcus jannaschii
. For the mid-log-to-early-stationary-phase transition of a
T. maritima
monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in
T. maritima
at this level (15.5% of the genome) for the same growth phase transition. Interspecies H
2
transfer resulted in three- to fivefold-higher
T. maritima
cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding β-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of β-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where
T. maritima
coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.
Mycobacterium aviumGenes Associated with the Ability To Form a Biofilm
Authors: Yamazaki Yoshitaka , Danelishvili Lia , Wu Martin , MacNab Molly , Bermudez Luiz E.
Published: January 2006

Mycobacterium aviumis widely distributed in the environment, and it is chiefly found in water and soil.M. avium, as well asMycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened anM. aviumgreen fluorescent protein (GFP) promoter library inM. smegmatisfor genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression ≥2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation inM. aviumwas confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed usingM. aviumstrain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues ofM. tuberculosis6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production ofM. aviumbiofilm.
Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of
Mycobacterium avium
subsp.
avium
and
Mycobacterium avium
subsp.
paratuberculosis
in Potable-Water Biofilms
Authors: Lehtola Markku J. , Torvinen Eila , Miettinen Ilkka T. , Keevil C. William
Published: January 2006

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting
Mycobacterium avium
bacteria, including the opportunistically pathogenic subspecies
M. avium
subsp.
avium
,
M. avium
subsp.
paratuberculosis
, and
M. avium
subsp.
silvaticum
, by the fluorescence in situ hybridization (FISH) method. There is evidence that
M. avium
subsp.
avium
especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only
M. avium
subsp.
avium
and
M. avium
subsp.
paratuberculosis
strains were hybridized. The PNA FISH method was applied successfully to detect
M. avium
subsp.
avium
spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.
Replication and Long-Term Persistence of Bovine and Human Strains of
Mycobacterium avium
subsp.
paratuberculosis
within
Acanthamoeba polyphaga
Authors: Mura Manuela , Bull Tim J. , Evans Hugh , Sidi-Boumedine Karim , McMinn Liz , Rhodes Glenn , Pickup Roger , Hermon-Taylor John
Published: January 2006

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens.
Mycobacterium avium
subsp.
paratuberculosis
is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with
M. avium
subsp.
paratuberculosis
is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of
Acanthamoeba polyphaga
with bovine and human strains of
M. avium
subsp.
paratuberculosis
. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of
M. avium
subsp.
paratuberculosis
, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of
A. polyphaga. M. avium
subsp.
paratuberculosis
detected by nested IS
900
PCR with amplicon sequencing and visualized by IS
900
in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative.
M. avium
subsp.
paratuberculosis
has the potential for long-term persistence in environmental protists.
Rapid Quantification of the Toxic Alga
Prymnesium parvum
in Natural Samples by Use of a Specific Monoclonal Antibody and Solid-Phase Cytometry
Authors: West N. J. , Bacchieri R. , Hansen G. , Tomas C. , Lebaron P. , Moreau H.
Published: January 2006

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga
Prymnesium parvum
in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several
P. parvum
strains and a
Prymnesium nemamethecum
strain but it did not cross-react with
P. parvum
strains from Scandinavia or any other algal strains, including species of the closely related genus
Chrysochromulina. Prymnesium
sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured
P. parvum
cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of
P. parvum
occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural
P. parvum
cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.
Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria
Authors: Bosma Tjibbe , Kanninga Rolf , Neef Jolanda , Audouy Sandrine A. L. , van Roosmalen Maarten L. , Steen Anton , Buist Girbe , Kok Jan , Kuipers Oscar P. , Robillard George , Leenhouts Kees
Published: January 2006

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the
Lactococcus lactis
peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium
L. lactis
, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.
Cellular Response of
Shewanella oneidensis
to Strontium Stress
Authors: Brown Steven D. , Martin Madhavi , Deshpande Sameer , Seal Sudipta , Huang Katherine , Alm Eric , Yang Yunfeng , Wu Liyou , Yan Tingfen , Liu Xueduan , Arkin Adam , Chourey Karuna , Zhou Jizhong , Thompson Dorothea K.
Published: January 2006

The physiology and transcriptome dynamics of the metal ion-reducing bacterium
Shewanella oneidensis
strain MR-1 in response to nonradioactive strontium (Sr) exposure were investigated. Studies indicated that MR-1 was able to grow aerobically in complex medium in the presence of 180 mM SrCl
2
but showed severe growth inhibition at levels above that concentration. Temporal gene expression profiles were generated from aerobically grown, mid-exponential-phase MR-1 cells shocked with 180 mM SrCl
2
and analyzed for significant differences in mRNA abundance with reference to data for nonstressed MR-1 cells. Genes with annotated functions in siderophore biosynthesis and iron transport were among the most highly induced (>100-fold [
P
< 0.05]) open reading frames in response to acute Sr stress, and a mutant (SO3032::pKNOCK) defective in siderophore production was found to be hypersensitive to SrCl
2
exposure, compared to parental and wild-type strains. Transcripts encoding multidrug and heavy metal efflux pumps, proteins involved in osmotic adaptation, sulfate ABC transporters, and assimilative sulfur metabolism enzymes also were differentially expressed following Sr exposure but at levels that were several orders of magnitude lower than those for iron transport genes. Precipitate formation was observed during aerobic growth of MR-1 in broth cultures amended with 50, 100, or 150 mM SrCl
2
but not in cultures of the SO3032::pKNOCK mutant or in the abiotic control. Chemical analysis of this precipitate using laser-induced breakdown spectroscopy and static secondary ion mass spectrometry indicated extracellular solid-phase sequestration of Sr, with at least a portion of the heavy metal associated with carbonate phases.
Role of Tryptophan Residues in Toxicity of Cry1Ab Toxin from
Bacillus thuringiensis
Authors: Padilla Cristopher , Pardo-López Liliana , de la Riva Gustavo , Gómez Isabel , Sánchez Jorge , Hernandez Georgina , Nuñez Maria Eugenia , Carey Marianne P. , Dean Donald H. , Alzate Oscar , Soberón Mario , Bravo Alejandra
Published: January 2006

Bacillus thuringiensis
produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against
Manduca sexta
. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.
Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria
Authors: Ferrari Belinda C. , Tujula Niina , Stoner Kate , Kjelleberg Staffan
Published: January 2006

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to be superior to conventional FISH for the detection of microcolonies that are cultivated by using the soil substrate membrane system. CARD-FISH enabled real-time identification of oligophilic microcolony-forming soil bacteria without the requirement for enrichment on complex media and the associated shifts in community composition.
The
Bifidobacterium longum
NCIMB 702259
T
ctr
Gene Codes for a Novel Cholate Transporter
Authors: Price Claire E. , Reid Sharon J. , Driessen Arnold J. M. , Abratt Valerie R.
Published: January 2006

Preexposure of
Bifidobacterium longum
NCIMB 702259
T
to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The
B. longum ctr
gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant
Escherichia coli
KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [
14
C]cholate.
Immobilization of Cells with Surface-Displayed Chitin-Binding Domain
Authors: Wang Jen-You , Chao Yun-Peng
Published: January 2006

To explore chitin-binding domain (ChBD)-based cell immobilization, a tripartite gene fusion consisting of an in-frame fusion of ChBD to
lpp
and
ompA
was constructed and expressed in
Escherichia coli
. ChBD-displayed cells exhibited highly specific and stable binding to chitin within a wide range of pHs (5 to 8) and temperatures (15 to 37°C). These results illustrate the promising use of this approach for engineering applications.
Naturally Transformable
Acinetobacter
sp. Strain ADP1 Belongs to the Newly Described Species
Acinetobacter baylyi
Authors: Vaneechoutte Mario , Young David M. , Ornston L. Nicholas , De Baere Thierry , Nemec Alexandr , Van Der Reijden Tanny , Carr Emma , Tjernberg Ingela , Dijkshoorn Lenie
Published: January 2006

Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable
Acinetobacter
sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species
Acinetobacter baylyi
. Transformation assays demonstrate that the
A. baylyi
type strain B2
T
and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus
Acinetobacter
, characterized by the high transformability of its strains that have been cultured thus far.
Autoinducer 2 Affects Biofilm Formation byBacillus cereus
Authors: Auger Sandrine , Krin Evelyne , Aymerich Stéphane , Gohar Michel
Published: January 2006

Cell-free supernatants from growingBacillus cereusstrain ATCC 10987 induced luminescence in aPhotorhabdus luminescensΔluxSmutant, indicating the production of functional autoinducer 2 (AI-2). The exogenous addition of in vitro synthesized AI-2 had an inhibitory effect on biofilm formation byB. cereusand promoted release of the cells from a preformed biofilm.
Multiphasic Approach To Study the Bacterial Ecology of Fermented Sausages Inoculated with a Commercial Starter Culture
Authors: Cocolin Luca , Urso Rosalinda , Rantsiou Kalliopi , Cantoni Carlo , Comi Giuseppe
Published: January 2006

In this paper, the ability of a commercial starter culture to perform a sausage fermentation is evaluated. Molecular analysis revealed the presence of several strains of the same species contained in the starter culture with different behavior during the fermentation, and the contribution of
Lactobacillus curvatus
, which was only marginally isolated during the transformation.
Butyrate Specifically Down-Regulates
Salmonella
Pathogenicity Island 1 Gene Expression
Authors: Gantois I. , Ducatelle R. , Pasmans F. , Haesebrouck F. , Hautefort I. , Thompson A. , Hinton J. C. , Van Immerseel F.
Published: January 2006

Invasion of intestinal epithelial cells by
Salmonella enterica
is decreased after exposure to butyric acid. To understand the molecular mechanisms of this phenomenon, a comparative transcriptomic analysis of
Salmonella enterica
serovar Enteritidis and
Salmonella enterica
serovar Typhimurium grown in medium supplemented with butyrate was performed. We found that butyrate down-regulated the expression of 19 genes common to both serovars by a factor of twofold or more, and 17 of these genes localized to the
Salmonella
pathogenicity island 1 (SPI1). These included the SPI1 regulatory genes
hilD
and
invF
. Of the remaining two genes,
ampH
has 91% homology to an
Escherichia coli
penicillin-binding protein and
sopE2
encodes a type III-secreted effector protein associated with invasion but located at a separate site on the chromosome from SPI1.
Propane and
n
-Butane Oxidation by
Pseudomonas putida
GPo1
Authors: Johnson Erika L. , Hyman Michael R.
Published: January 2006

Propane and
n
-butane inhibit methyl
tertiary
butyl ether oxidation by
n-
alkane-grown
Pseudomonas putida
GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of
n-
octane.
HacA-Independent Induction of Chaperone-Encoding Gene
bipA
in
Aspergillus niger
Strains Overproducing Membrane Proteins
Authors: Davé Anoushka , Jeenes David J. , Mackenzie Donald A. , Archer David B.
Published: January 2006

Transcription of two unfolded protein response genes,
hacA
and
bipA
, was examined in
Aspergillus niger
strains overproducing membrane proteins. Despite elevated
bipA
mRNA levels, no 5′-truncated
hacA
transcript was detected, raising the possibility of a
hacA
-independent induction of
bipA
mRNA under the stress of membrane protein overproduction in
A. niger
.
Emergence of Biofilm-Forming Subpopulations upon Exposure of
Escherichia coli
to Environmental Bacteriophages
Authors: Lacqua Andrea , Wanner Oskar , Colangelo Teresa , Martinotti Maria Giovanna , Landini Paolo
Published: January 2006

Exposure of
Escherichia coli
MG1655 to environmental bacteriophages results in rapid selection for phage-tolerant subpopulations displaying increased biofilm formation. Analysis of one phage-tolerant strain revealed large amounts of the DNA-binding Dps protein in the outer membrane protein and production of fimbria-like structures. In
dps
and
fimA
mutant derivatives of MG1655, no selection of phage-tolerant bacteria upon exposure to bacteriophages occurred, suggesting a role for Dps and type I pili in bacteriophage tolerance.
Microbial Conversion of Glycerol to 1,3-Propanediol: Physiological Comparison of a Natural Producer,
Clostridium butyricum
VPI 3266, and an Engineered Strain,
Clostridium acetobutylicum
DG1(pSPD5)
Authors: González-Pajuelo María , Meynial-Salles Isabelle , Mendes Filipa , Soucaille Philippe , Vasconcelos Isabel
Published: January 2006

Clostridium acetobutylicum
is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from
C. butyricum
VPI 3266, was introduced into
C. acetobutylicum
DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant
C. acetobutylicum
DG1(pSPD5) strain with that of the natural 1,3-propanediol producer
C. butyricum
VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same “global behavior” was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH
2
flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different:
C. butyricum
uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while
C. acetobutylicum
uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in
C. butyricum
VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in
C. acetobutylicum
DG1(pSPD5), and (ii) while the ferredoxin-NAD
+
reductase activity is high and the NADH-ferredoxin reductase activity is low in
C. acetobutylicum
DG1(pSPD5), the reverse is observed for
C. butyricum
VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the
C. acetobutylicum
DG1(pSPD5) culture, explaining why this microorganism produces lactate.
Presence of
Salmonella
and
Campylobacter
spp. in Wild Small Mammals on Organic Farms
Authors: Meerburg B. G. , Jacobs-Reitsma W. F. , Wagenaar J. A. , Kijlstra A.
Published: January 2006

The presence of
Salmonella
and
Campylobacter
spp. in rodents and insectivores (
n
= 282) was investigated on organic farms. Infections were encountered in house mice (8 of 83
Campylobacter
positive and 1 of 83
Salmonella
sp. strain Livingstone positive) and brown rats (1 of 8
Campylobacter
positive) but not in other species. No shared
Campylobacter
genotypes were found between rodent and pig manure isolates. Effective on-farm rodent management is recommended.
Expression of the
ubiE
Gene of
Geobacillus stearothermophilus
V in
Escherichia coli
K-12 Mediates the Evolution of Selenium Compounds into the Headspace of Selenite- and Selenate-Amended Cultures
Authors: Swearingen J. W. , Fuentes D. E. , Araya M. A. , Plishker M. F. , Saavedra C. P. , Chasteen T. G. , Vásquez C. C.
Published: January 2006

The
ubiE
gene of
Geobacillus stearothermophilus
V, with its own promoter, was cloned and introduced into
Escherichia coli
. The cloned gene complemented the
ubiE
gene deficiency of
E. coli
AN70. In addition, the expression of this gene in
E. coli
JM109 resulted in the evolution of volatile selenium compounds when these cells were grown in selenite- or selenate-amended media. These compounds were dimethyl selenide and dimethyl diselenide.
Characterization of a Novel PepF-Like Oligopeptidase Secreted by
Bacillus amyloliquefaciens
23-7A
Authors: Chao Shiou-Huei , Cheng Tzu-Hao , Shaw Chin-Ying , Lee Meng-Hwan , Hsu Yuan-Hsun , Tsai Ying-Chieh
Published: January 2006

An oligopeptidase from
Bacillus amyloliquefaciens
23-7A was characterized along with its biochemical activities and structural gene. The protein's amino acid sequence and enzymatic activities were similar to those of other bacterial PepFs, which belong to metallopeptidase family M3. While most bacterial PepFs are cytoplasmic endopeptidases, the identified PepF
Ba
oligopeptidase is a secreted protein and may facilitate the process of sporulation.
Bilirubin Oxidase Activity of
Bacillus subtilis
CotA
Authors: Sakasegawa Shin-ichi , Ishikawa Hidehiko , Imamura Shigeyuki , Sakuraba Haruhiko , Goda Shuichiro , Ohshima Toshihisa
Published: January 2006

The spore coat protein CotA from
Bacillus subtilis
was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.
Prevalence of
Borrelia burgdorferi
Sensu Lato in Ticks Collected from Migratory Birds in Switzerland
Authors: Poupon Marie-Angèle , Lommano Elena , Humair Pierre-François , Douet Véronique , Rais Olivier , Schaad Michael , Jenni Lukas , Gern Lise
Published: January 2006

The prevalence of ticks infected by
Borrelia burgdorferi
sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation.
Ixodes ricinus
was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively.
Borrelia valaisiana
was the species detected most frequently in ticks, followed by
Borrelia garinii
and
Borrelia lusitaniae
. Among birds infested by infected ticks, 23% (6/26) were infested by
B. lusitaniae
-infected larvae. Migratory birds appear to be reservoir hosts for
B. lusitaniae
.
Ultrasensitive Human Immunodeficiency Virus Type 1 p24 Antigen Assay Modified for Use on Dried Whole-Blood Spots as a Reliable, Affordable Test for Infant Diagnosis
Authors: Patton Janet C. , Sherman Gayle G. , Coovadia Ashraf H. , Stevens Wendy S. , Meyers Tammy M.
Published: January 2006

The ultrasensitive human immunodeficiency virus (HIV) p24 antigen assay was modified for use on pediatric dried whole-blood spots on Whatman no. 1 filter paper. The modified assay was found to be reliable and accurate, making it an affordable tool for pediatric HIV diagnosis in developing countries.
Can the Level of Immunosuppression in Human Immunodeficiency Virus-Infected Patients Affect the Reliability of Human T-Cell Lymphotropic Virus Type 2 Serological Diagnosis?
Authors: Bassani Sylvina , Toro Carlos , Jiménez Victoria , Rodés Berta , Soriano Vincent
Published: January 2006

A total of 175 human immunodeficiency virus (HIV)-positive intravenous drug users (IDU) with CD4 cell counts of <200 cells/μl were matched with 175 HIV-positive IDU with CD4 cell counts of >500 cells/μl. Enzyme immunoassay (EIA) reactivity and human T-cell lymphotropic virus type 2 (HTLV-2) Western blot (WB) positivity were more frequently observed in subjects with CD4 cell counts of >500 cells/μl. Most of the subjects with low CD4 cell counts and EIA reactivity carried HTLV-2 infection (WB positive and/or PCR positive). No subjects with low CD4 cell counts and a lack of reactive EIA were PCR positive for HTLV-2. Therefore, a negative EIA result can confidently discharge HTLV-2 infection in HIV-infected patients with severe immunosuppression, whereas PCR should be performed for subjects with a reactive HTLV EIA which is not further confirmed by WB.
Diagnostic Implications of Antigen-Induced Gamma Interferon Production by Blood Leukocytes from
Mycobacterium bovis
-Infected Reindeer (
Rangifer tarandus
)
Authors: Waters W. R. , Palmer M. V. , Slaughter R. E. , Jones S. L. , Pitzer J. E. , Minion F. C.
Published: January 2006

The only approved method of tuberculosis (TB) surveillance of reindeer within the United States is tuberculin skin testing; however, skin testing has an apparent lack of specificity, since numerous reindeer are classified as reactors, yet
Mycobacterium bovis
is not isolated from tissues upon necropsy. The objective of this study was to evaluate the ability of an in vitro assay (the Cervigam assay) to detect gamma interferon (IFN-γ) produced by blood leukocytes in response to mycobacterial antigens from
M. bovis
-infected reindeer. Thirteen male reindeer ∼9 months of age were inoculated with 10
5
CFU
M. bovis
in their tonsillar crypts. Stimulation of whole-blood cultures with a mitogen resulted in significant production of IFN-γ compared to that by nonstimulated samples. Responses by infected reindeer to
M. bovis
purified protein derivative (PPD) were as much as 3.5-fold higher than those by noninfected reindeer (
n
= 4). Despite differences in responses to PPD by the two groups, reindeer within the noninfected group had responses of >0.1 change in optical density (ΔOD) (a level generally considered positive) to PPD. Mean responses by infected reindeer to a rESAT-6-CFP-10 fusion protein (
Mycobacterium tuberculosis
complex specific) were as much as 20-fold higher than respective responses by noninfected reindeer at all time points. Additionally, responses by 3/4 noninfected reindeer were <0.1 ΔOD (considered negative) at each time point. To further evaluate the specificity of the assay, samples were collected from reindeer in a TB-free herd. All reindeer had responses to mitogen; however, only 1 of 38 had a response to PPD, and none of the reindeer responded to rESAT-6-CFP-10. Together, these findings indicate that IFN-γ-based tests may prove useful for TB surveillance of reindeer.
Development of a Clinical Assay To Evaluate Toll-Like Receptor Function
Authors: Deering Raquel P. , Orange Jordan S.
Published: January 2006

Toll-like receptors (TLRS) recognize pathogen-associated molecular patterns to enable innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR function in order to evaluate patients with recurrent infection who would be suspected of having a genetic defect affecting TLR signaling. We chose to study peripheral blood mononuclear cells (PBMCS) to avoid potential influences of soluble factors contained in whole blood, and we utilized ligands for TLRS 1/2, 2/6, 3, 4, 5, 6, 7, and 9. Tumor necrosis factor (TNF) production in PBMC supernatants was measured by an enzyme-linked immunosorbent assay after TLR ligand stimulation and was dependent on gene transcription and NF-κB activation. Some variables affecting the assay were assessed, including the effects of: blood anticoagulant, serum-containing media, incubation time, ligand storage, blood storage time, and cell cryopreservation. By using optimized assay conditions, effective concentrations of individual ligands and mean responses to those ligands were established for healthy control donors. Finally, three patients with a mutation in theIKBKGgene, encoding the NF-κB essential modulator (NEMO) protein, were evaluated as disease controls and were almost uniformly below the standard deviation of healthy donors for all ligands tested. Although a number of variables influence TLR ligand-induced TNF responses, this assay can be optimized for potential clinical use to screen patients with primary immunodeficiencies affecting TLR function.
Generation of Antibodies against Bovine Recombinant Prion Protein in Various Strains of Mice
Authors: Andrievskaia Olga , McRae Heather , Elmgren Cathy , Huang Hongsheng , Balachandran Aru , Nielsen Klaus
Published: January 2006

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, belong to a group of neurodegenerative disorders affecting humans and animals. To date, definite diagnosis of prion disease can only be made by analysis of tissue samples for the presence of protease-resistant misfolded prion protein (PrP
Sc
). Monoclonal antibodies (MAbs) to the prion protein provide valuable tools for TSE diagnosis, as well as for basic research on these diseases. In this communication, the development of antibodies against recombinant bovine prion protein (brecPrP) in four strains of mice (BALB/c, ND4, SJL, and NZB/NZW F
1
) is described. Immunization of autoimmunity-prone NZB/NZW F
1
and SJL mice with brecPrP was applied to overcome self-tolerance against the prion protein. ND4 and SJL mice did not develop an immune response to brecPrP. BALB/c mice produced antibody titers of 1:1,000 to 1:1,500 in an enzyme-linked immunosorbent assay (ELISA), while NZB/NZW F
1
mice responded with titers of 1:7,000 to 1:11,000. A panel of 71 anti-brecPrP MAbs recognizing continuous and discontinuous epitopes was established from BALB/c and NZB/NZW F
1
mice. Seven anti-brecPrP MAbs reacted with both the cellular form of PrP and protease K-resistant PrP
Sc
from sheep brain in Western blot assays. The epitope specificity of these MAbs was determined, and applicability to immunohistochemical detection of prions was studied. The MAbs generated will be useful tools in the development of TSE immunochemical diagnosis and for research. This is the first report of the development of anti-PrP MAbs by use of autoimmune NZB/NZW F
1
mice as an alternative approach for the generation of PrP-specific MAbs.
Seroepidemiology ofKlebsiella pneumoniaein an Australian Tertiary Hospital and Its Implications for Vaccine Development
Authors: Jenney Adam W. , Clements Abigail , Farn Jacinta L. , Wijburg Odilia L. , McGlinchey Andrew , Spelman Denis W. , Pitt Tyrone L. , Kaufmann Mary E. , Liolios Lisa , Moloney Margaret B. , Wesselingh Steven L. , Strugnell Richard A.
Published: January 2006

The aim of this study was to determine the diversity ofKlebsiella pneumoniaecapsular serotypes in an Australian setting. Consecutive (n= 293) nonrepetitive isolates ofK. pneumoniaefrom a large teaching hospital laboratory were analyzed. The majority of isolates were from urinary specimens (60.8%); the next most common source was sputum (14.3%), followed by blood (14%). Serotyping revealed a wide range of capsule types. K54 (17.1%), K28 (4.1%), and K17 (3.1%) were the most common, and K54 isolates displayed a high degree of clonality, suggesting a common, nosocomial source. In vitro, one K54 isolate was more adherent to urinary catheters and HEp-2 cells than four other tested isolates; it was slightly more resistant to chlorhexidine but was more susceptible to drying than heavily encapsulated strains. This is the first seroprevalence survey ofK. pneumoniaeto be performed on Australian isolates, and the high level of diversity of serotypes suggests that capsule-based immunoprophylaxis might not be useful for Australia. In addition there are significant differences in the predominance of specific serotypes compared to the results of surveys performed overseas, which has important implications for capsule-based immunoprophylaxis aimed at a global market.
Characterization of a Strain of Community-AssociatedMethicillin-Resistant
Staphylococcus aureus
WidelyDisseminated in the UnitedStates
Authors: Tenover Fred C. , McDougal Linda K. , Goering Richard V. , Killgore George , Projan Steven J. , Patel Jean B. , Dunman Paul M.
Published: January 2006

A
highly stable strain of
Staphylococcus aureus
with a
pulsed-field gel electrophoresis type of USA300 and multilocus sequence
type 8 has been isolated from patients residing in diverse geographic
regions of the United States. This strain, designated USA300-0114, is a
major cause of skin and soft tissue infections among persons in
community settings, including day care centers and correctional
facilities, and among sports teams, Native Americans, men who have sex
with men, and military recruits. The organism is typically resistant to
penicillin, oxacillin, and erythromycin (the latter mediated by
msrA
) and carries SCC
mec
type IVa. This strain is
variably resistant to tetracycline [mediated by
tet
(K)];
several recent isolates have decreased susceptibility to
fluoroquinolones.
S. aureus
USA300-0114 harbors the genes
encoding the Panton-Valentine leucocidin toxin. DNA sequence analysis
of the direct repeat units within the
mec
determinant of 30
USA300-0114 isolates revealed differences in only a single isolate.
Plasmid analysis identified a common 30-kb plasmid that hybridized with
blaZ
and
msrA
probes and a 3.1-kb cryptic plasmid. A
4.3-kb plasmid encoding
tet
(K) and a 2.6-kb plasmid encoding
ermC
were observed in a few isolates. DNA microarray analysis
was used to determine the genetic loci for a series of virulence
factors and genes associated with antimicrobial resistance. Comparative
genomics between USA300-0114 and three other
S. aureus
lineages (USA100, USA400, and USA500) defined a set of
USA300-0114-specific genes, which may facilitate the strain's
pathogenesis within diverse
environments.
Faster Identification of Pathogens in Positive Blood Cultures by Fluorescence In Situ Hybridization in Routine Practice
Authors: Peters Remco P. H. , Savelkoul Paul H. M. , Simoons-Smit Alberdina M. , Danner Sven A. , Vandenbroucke-Grauls Christina M. J. E. , van Agtmael Michiel A.
Published: January 2006

Rapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice. After Gram staining, blood culture fluids were simultaneously further identified with FISH and with conventional culture methods. Results and points in time of FISH and culture identification (provisional and final identifications) were collected and compared. For 91% of microorganisms, the genus or family name was identified, and for 79%, the species name could be attributed. The sensitivity and specificity of the individual probes exceeded 95%, except for the
Enterobacteriaceae
probe (sensitivity, 89%). Cross-hybridization was obtained with the
Klebsiella pneumoniae
probe for
Klebsiella oxytoca
. The time gains of FISH and final culture identification were more than 18 h for bacteria and 42 h for yeasts. With FISH,
Staphylococcus aureus
was differentiated from coagulase-negative staphylococci 1.4 h faster than by provisional identification (
P
< 0.001). In conclusion, FISH allows rapid and reliable identification of the majority of microorganisms in growth-positive blood cultures. The substantial time gain of identification with FISH may allow same-day adjustment of antimicrobial therapy, and FISH is especially useful if no provisional identification is obtained. With further extension of the number of probes and a reduction in turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections.