bluuebunny/crossref_metadata_2025 huggingface dataset:
Real-Time Quantitative Reverse Transcription-PCR Analysis of Expression Stability ofActinobacillus pleuropneumoniaeHousekeeping Genes during In Vitro Growth under Iron-Depleted Conditions
Authors: Nielsen K. Klitgaard , Boye M.
Published: June 2005

The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogenActinobacillus pleuropneumoniaeby real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls, as such controls have not been defined yet for this bacterium. Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. First, the stability of expression of five genes, theglyA,tpiA,pykA,recF, andrhoAPgenes involved in basic housekeeping, was evaluated on the basis of the mean pairwise variation. All the housekeeping genes included were stably expressed under the conditions investigated and consequently were included in the normalization procedure. Next, the geometric mean of the internal control genes was used to correct five genes of interest. These genes were three genes involved in iron acquisition (tbpA, exbB, andfhuD), the heat shock protein genegroEL, and a putative quorum-sensing gene (luxS). The level oftbpA, exbB, andfhuDexpression inA. pleuropneumoniaeshowed significant up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT-PCR, with theglyA,tpiA,pykA,recF, andrhoAPgenes as internal controls, for future similar gene expression studies inA. pleuropneumoniae.
Indications for Acquisition of Reductive Dehalogenase Genes through Horizontal Gene Transfer byDehalococcoides ethenogenesStrain 195
Authors: Regeard Christophe , Maillard Julien , Dufraigne Christine , Deschavanne Patrick , Holliger Christof
Published: June 2005

The genome ofDehalococcoides ethenogenesstrain 195, an anaerobic dehalorespiring bacterium, contains 18 copies of putative reductive dehalogenase genes, including the well-characterizedtceAgene, whose gene product functions as the key enzyme in the environmentally important dehalorespiration process. The genome ofD. ethenogeneswas analyzed using a bioinformatic tool based on the frequency of oligonucleotides. The results in the form of a genomic signature revealed several local disruptions of the host signature along the genome sequence. These fractures represent DNA segments of potentially foreign origin, so-called atypical regions, which may have been acquired by an ancestor through horizontal gene transfer. Most interestingly, 15 of the 18 reductive dehalogenase genes, including thetceAgene, were found to be located in these regions, strongly indicating the foreign nature of the dehalorespiration activity. The GC content and the presence of recombinase genes within some of these regions corroborate this hypothesis. A hierarchical classification of the atypical regions containing the reductive dehalogenase genes indicated that these regions were probably acquired by several gene transfer events.
Metabolic Engineering of the Phenylpropanoid Pathway in
Saccharomyces cerevisiae
Authors: Jiang Hanxiao , Wood Karl V. , Morgan John A.
Published: June 2005

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (
PAL
) from
Rhodosporidium toruloides
, 4-coumarate:coenzyme A (CoA) ligase (
4CL
) from
Arabidopsis thaliana
, and chalcone synthase (
CHS
) from
Hypericum androsaemum
into two
Saccharomyces cerevisiae
strains, namely, AH22 and a
pad1
knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual
GAL10
promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both
trans
-cinnamic acid and
p
-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter
−1
of naringenin and 0.8 mg liter
−1
of pinocembrin. Several by-products, such as 2′,4′,6′-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor
l
-tyrosine.
Biomass Production and Assimilation of Dissolved Organic Matter by SAR11 Bacteria in the Northwest Atlantic Ocean
Authors: Malmstrom Rex R. , Cottrell Matthew T. , Elifantz Hila , Kirchman David L.
Published: June 2005

Members of the SAR11 clade often dominate the composition of marine microbial communities, yet their contribution to biomass production and the flux of dissolved organic matter (DOM) is unclear. In addition, little is known about the specific components of the DOM pool utilized by SAR11 bacteria. To better understand the role of SAR11 bacteria in the flux of DOM, we examined the assimilation of leucine (a measure of biomass production), as well as free amino acids, protein, and glucose, by SAR11 bacteria in the Northwest Atlantic Ocean. We found that when SAR11 bacteria were >25% of total prokaryotes, they accounted for about 30 to 50% of leucine incorporation, suggesting that SAR11 bacteria were major contributors to bacterial biomass production and the DOM flux. Specific growth rates of SAR11 bacteria either equaled or exceeded growth rates for the total prokaryotic community. In addition, SAR11 bacteria were typically responsible for a greater portion of amino acid assimilation (34 to 61%) and glucose assimilation (45 to 57%) than of protein assimilation (≤34%). These data suggest that SAR11 bacteria do not utilize various components of the DOM pool equally and may be more important to the flux of low-molecular-weight monomers than to that of high-molecular-weight polymers.
Molecular and Phenotypic Analyses Reveal Association of Diverse
Colletotrichum acutatum
Groups and a Low Level of
C. gloeosporioides
with Olive Anthracnose
Authors: Talhinhas Pedro , Sreenivasaprasad S. , Neves-Martins João , Oliveira Helena
Published: June 2005

Anthracnose (
Colletotrichum
spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of
Colletotrichum
spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128
Colletotrichum
spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and β-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed
Colletotrichum acutatum
to be dominant (>97%) with limited occurrence of
Colletotrichum gloeosporioides
(<3%). Among
C. acutatum
populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other
C. acutatum
groups and
C. gloeosporioides
were sporadic in their occurrence, mostly related to marginal areas of olive cultivation.
C. gloeosporioides
, isolated from olive fruits with symptoms indistinguishable from those of
C. acutatum
, showed same virulence rating as the most virulent
C. acutatum
isolate from group A2.
C. acutatum
and
C. gloeosporioides
isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from β-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of
C. acutatum
groups.
Aspergillus parasiticus
Cyclase Catalyzes Two Dehydration Steps in Aflatoxin Biosynthesis
Authors: Sakuno Emi , Wen Ying , Hatabayashi Hidemi , Arai Hatsue , Aoki Chiemi , Yabe Kimiko , Nakajima Hiromitsu
Published: June 2005

In the aflatoxin biosynthetic pathway, 5′-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2′
S
,5′
S
)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol.
69:
6418-6426, 2003). Interestingly, the
N
-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (
vbs
). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of
Aspergillus parasiticus
, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in
Pichia pastoris
, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of
A. parasiticus
SYS-4 (= NRRL 2999) with
vbs
deleted accumulated large amounts of OAVN, 5′-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the
vbs
gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.
Identification of Endopeptidase Genes from the Genomic Sequence of
Lactobacillus helveticus
CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides
Authors: Sridhar Vidya R. , Hughes Joanne E. , Welker Dennis L. , Broadbent Jeffery R. , Steele James L.
Published: June 2005

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of
Lactobacillus helveticus
CNRZ32 and designated
pepO3
,
pepF
, and
pepE2
. The ability of cell extracts from
Escherichia coli
DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and α
S1
-casein (α
S1
-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards α
S1
-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in
Lactococcus lactis
, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the
pepO3
promoter. Cell extracts of
L. lactis
derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and α
S1
-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides α
S1
-CN (f1-9), α
S1
-CN (f1-13), and α
S1
-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and α
S1
-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that
L. helveticus
CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.
Genetic Analysis of Biosurfactant Production inUstilago maydis
Authors: Hewald Sandra , Josephs Katharina , Bölker Michael
Published: June 2005

The dimorphic basidiomyceteUstilago maydisproduces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated β-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes,emt1andcyp1, that are essential for the production of fungal extracellular glycolipids.emt1is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the genecyp1resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.
Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars
Authors: Corcoran B. M. , Stanton C. , Fitzgerald G. F. , Ross R. P.
Published: June 2005

Lactobacillus rhamnosusGG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose onL. rhamnosusGG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log10-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with otherLactobacillusstrains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhancedL. rhamnosusGG survival from 6.4 to 8 log10CFU ml−1in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition ofN′,N′-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F0F1-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F0F1-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 × 106-fold compared with the survival of the wild type (which had a viability of 8.02 log10CFU ml−1).L. rhamnosusGG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log10CFU ml−1in the presence of glucose. The data indicate that glucose provides ATP to F0F1-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.
Molecular Characterization of Plasmid pBM300 from
Bacillus megaterium
QM B1551
Authors: Kunnimalaiyaan Muthusamy , Vary Patricia S.
Published: June 2005

Strain QM B1551 of
Bacillus megaterium
contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea.
oriT
and
ssoT-
like regions were detected near two
mob
genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.
Use of a Tetracycline-Inducible System for Conditional Expression in
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
Authors: Carroll Paul , Muttucumaru D. G. Niranjala , Parish Tanya
Published: June 2005

A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in
M. smegmatis
and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in
M. tuberculosis
, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of
M. tuberculosis
that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that
trpD
was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of
M. tuberculosis
. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.
Increase in Xylanase Production by
Streptomyces lividans
through Simultaneous Use of the Sec- and Tat-Dependent Protein Export Systems
Authors: Gauthier Céline , Li Haiming , Morosoli Rolf
Published: June 2005

Xylanase B1 (XlnB1) from
Streptomyces lividans
is a protein consisting of two discrete structural and functional units, an N-terminal catalytic domain and a C-terminal substrate binding domain. In the culture medium, two forms of xylanase B are present, namely, XlnB1 and XlnB2, the latter of which corresponds to the catalytic domain of XlnB1 deprived of the substrate binding domain. Both forms of the xylanase have the same activity on xylan. The enzyme is secreted through the Sec-dependent pathway with a better yield of XlnB1 than XlnB2. Interestingly, XlnB2 exhibits 80% identity with XlnC which is secreted exclusively through the Tat-dependent pathway. To demonstrate whether XlnB1 and XlnB2 could also be secreted through the Tat-dependent pathway, the Tat-targeting
xlnC
signal sequence was fused to the structural genes of
xlnB1
and
xlnB2
. Both XlnB1 and XlnB2 were secreted through the Tat-dependent pathway, but XlnB2 was better produced than XlnB1. As XlnB1 and XlnB2 could be better secreted through the Sec- and Tat-dependent systems, respectively, a copy of the structural gene of
xlnB1
fused to a Sec signal sequence and a copy of the structural gene of
xlnB2
fused to a Tat signal sequence were inserted into the same plasmid under the control of the
xlnA
promoter. The transformant produced xylanase activity which corresponded approximately to the sum of activities of the individual strain producing xylanase by either the Sec- or Tat-dependent secretion system. This indicated that both secretion systems are functional and independent of each other in the recombinant strain. This is the first report on the efficient secretion of a protein using two different secretion systems at the same time. Assuming that the protein to be secreted could be properly folded prior to and after translocation via the Tat- and Sec-dependent pathways, respectively, the simultaneous use of the Sec- and Tat-dependent pathways provides an efficient means to increase the production of a given protein.
Influence of Growth Phase on Adhesion Kinetics of
Escherichia coli
D21g
Authors: Walker Sharon L. , Hill Jane E. , Redman Jeremy A. , Elimelech Menachem
Published: June 2005

The influence of bacterial growth stage and the evolution of surface macromolecules on cell adhesion have been examined by using a mutant of
Escherichia coli
K-12. To better understand the adhesion kinetics of bacteria in the mid-exponential and stationary growth phases under flow conditions, deposition experiments were conducted in a well-controlled radial stagnation point flow (RSPF) system. Complementary cell characterization techniques were conducted in combination with the RSPF experiments to evaluate the hydrophobicity, electrophoretic mobility, size, and titratable surface charge of the cells in the two growth phases considered. It was observed that cells in stationary phase were notably more adhesive than those in mid-exponential phase. This behavior is attributed to the high degree of local charge heterogeneity on the outer membranes of stationary-phase cells, which results in decreased electrostatic repulsion between the cells and a quartz surface. The mid-exponential-phase cells, on the other hand, have a more uniform charge distribution on the outer membrane, resulting in greater electrostatic repulsion and, subsequently, less adhesion. Our results suggest that the macromolecules responsible for this phenomenon are outer membrane-bound proteins and lipopolysaccharide-associated functional groups.
Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide
Authors: Thurston-Enriquez Jeanette A. , Haas Charles N. , Jacangelo Joseph , Gerba Charles P.
Published: June 2005

Chlorine dioxide (ClO
2
) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions.
Ct
values (the concentration of ClO
2
multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40
Ct
ranges for 4-log inactivation (
Ct
99.99%
) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min
Ct
99.99%
ranges were observed for pH 6 and 8, respectively. FCV
Ct
99.99%
ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments,
Ct
99.99%
ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of
Ct
values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale
Ct
99.99%
ranges and EFH model
Ct
99.99%
values demonstrated that FCV is more resistant to ClO
2
than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual
Ct
99.99%
values are higher than
Ct
99.99%
values calculated from bench-scale experiments and from EFH model application.
Abundant Respirable Ergot Alkaloids from the Common Airborne Fungus
Aspergillus fumigatus
Authors: Panaccione Daniel G. , Coyle Christine M.
Published: June 2005

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals.
Aspergillus fumigatus
, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if
A. fumigatus
accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of
A. fumigatus
. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for
A. fumigatus
than for
Aspergillus nidulans
,
Aspergillus niger
, and
Stachybotrys chartarum
. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of
A. fumigatus
presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.
Abundance and Diversity of Viruses in Six Delaware Soils
Authors: Williamson Kurt E. , Radosevich Mark , Wommack K. Eric
Published: June 2005

The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 × 10
9
to 4.17 × 10
9
g
−1
dry weight) than in agricultural soils (8.7 × 10
8
to 1.1 × 10
9
g
−1
dry weight). Soil VLP abundance was significantly correlated to moisture content (
r
= 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (
r
= 0.885) and viral (
r
= 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.
Genomic Islands in theCorynebacterium efficiensGenome
Authors: Zhang Ren , Zhang Chun-Ting
Published: June 2005

Corynebacterium efficiensis a gram-positive nonpathogenic bacterium which can grow and produce glutamate at 40°C or above. By using the cumulative GC profile method, we have identified four genomic islands which have many unifying genomic island-specific features in theC. efficiensgenome. The presence of the gene encoding an aspartate kinase in a genomic island helps explain the unexpected low thermal stability of this enzyme; i.e., the adaptive mutations have not occurred extensively due to the recent horizontal gene transfer.
Quantitative Real-Time PCR Assays for Detection of Human Adenoviruses and Identification of Serotypes 40 and 41
Authors: Jothikumar Narayanan , Cromeans Theresa L. , Hill Vincent R. , Lu Xiaoyan , Sobsey Mark D. , Erdman Dean D.
Published: June 2005

A quantitative real-time TaqMan PCR assay for detection of human adenoviruses (HAdV) was developed using broadly reactive consensus primers and a TaqMan probe targeting a conserved region of the hexon gene. The TaqMan assay correctly identified 56 representative adenovirus prototype strains and field isolates from all six adenovirus species (A to F). Based on infectious units, the TaqMan assay was able to detect as few as 0.4 and 0.004 infectious units of adenovirus serotype 2 (AdV2) and AdV41, respectively, with results obtained in less than 90 min. Using genomic equivalents, the broadly reactive TaqMan assay was able to detect 5 copies of AdV40 (which had zero mismatches with the PCR primers and probe), 8 copies of AdV41, and 350 copies of AdV3 (which had the most mismatches [seven] of any adenovirus serotype tested). For specific detection and identification of F species serotypes AdV40 and AdV41, a second real-time PCR assay was developed using fluorescence resonance energy transfer (FRET) probes that target the adenovirus fiber gene. The FRET-based assay had a detection limit of 3 to 5 copies of AdV40 and AdV41 standard DNA and was able to distinguish between AdV40 and AdV41 based on melting curve analysis. Both the TaqMan and FRET PCR assays were quantitative over a wide range of virus titers. Application of these assays for detection of adenoviruses and type-specific identification of AdV40 and AdV41 will be useful for identifying these viruses in environmental and clinical samples.
Continuous-Flow Capillary Assay for Measuring Bacterial Chemotaxis
Authors: Law Aaron M. J. , Aitken Michael D.
Published: June 2005

Bacterial chemotaxis may have a significant impact on the structure and function of bacterial communities. Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the bacterial transport parameters used in predictive models of chemotaxis. When the chemotactic bacteria consume the chemoeffector, the chemoeffector gradient to which the bacteria respond may be significantly perturbed by the consumption. Therefore, consumption of the chemoeffector can confound chemotaxis measurements if it is not accounted for. Current methods of quantifying chemotaxis use bacterial concentrations that are too high to preclude chemoeffector consumption or involve ill-defined conditions that make quantifying chemotaxis difficult. We developed a method of quantifying bacterial chemotaxis at low cell concentrations (∼10
5
CFU/ml), so metabolism of the chemoeffector is minimized. The method facilitates quantification of bacterial-transport parameters by providing well-defined boundary conditions and can be used with volatile and semivolatile chemoeffectors.
Amplified Intergenic Locus Polymorphism as a Basis for Bacterial Typing of
Listeria
spp. and
Escherichia
coli
Authors: Somer Lilach , Danin-Poleg Yael , Diamant Eran , Gur-Arie Riva , Palti Yniv , Kashi Yechezkel
Published: June 2005

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of
Listeria
monocytogenes
and 6 strains of
Listeria
spp. and 30 AILP markers among 27 strains of
Escherichia
coli
. Up to four alleles per locus were identified among
Listeria
strains, and up to six were identified among
E. coli
strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among
Listeria
and
E. coli
strains. A clear distinction between
L. monocytogenes
and
Listeria
spp. was demonstrated. In addition, the method separated
L. monocytogenes
into the three known lineages and discriminated the most common virulent serotypic group, 4b. In
E. coli
, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both
Listeria
and
E. coli
are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.
First Detection and Genotyping of Human-Associated Microsporidia in Pigeons from Urban Parks
Authors: Haro M. , Izquierdo F. , Henriques-Gil N. , Andrés I. , Alonso F. , Fenoy S. , del Águila C.
Published: June 2005

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for onlyEnterocytozoon bieneusi(9.7%), 5 forEncephalitozoon intestinalis(4%), and one forEncephalitozoon hellem(0.8%). Coinfections were detected in eight additional pigeons:E. bieneusiandE. hellemwere detected in six animals (4.8%);E. bieneusiwas associated withE. intestinalisin one case (0.8%); andE. hellemandE. intestinaliscoexisted in one pigeon. No positive samples forEncephalitozoon cuniculiwere detected. The internally transcribed spacer genotype could be completed for oneE. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in anE. hellemSpanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans forE. intestinalisandE. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.
Novel Putative Galactose Operon Involving Lacto-N-Biose Phosphorylase inBifidobacterium longum
Authors: Kitaoka Motomitsu , Tian Jiesheng , Nishimoto Mamoru
Published: June 2005

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract ofBifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein ofBifidobacterium longumNCC2705 encoded by the BL1641 gene. The homologous gene of the type strainB. longumJCM1217,lnpA, was expressed inEscherichia colito confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. ThelnpAgene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.
Validity of the Indicator Organism Paradigm for Pathogen Reduction in Reclaimed Water and Public Health Protection
Authors: Harwood Valerie J. , Levine Audrey D. , Scott Troy M. , Chivukula Vasanta , Lukasik Jerzy , Farrah Samuel R. , Rose Joan B.
Published: June 2005

The validity of using indicator organisms (total and fecal coliforms, enterococci,
Clostridium perfringens
, and F-specific coliphages) to predict the presence or absence of pathogens (infectious enteric viruses,
Cryptosporidium
, and
Giardia
) was tested at six wastewater reclamation facilities. Multiple samplings conducted at each facility over a 1-year period. Larger sample volumes for indicators (0.2 to 0.4 liters) and pathogens (30 to 100 liters) resulted in more sensitive detection limits than are typical of routine monitoring. Microorganisms were detected in disinfected effluent samples at the following frequencies: total coliforms, 63%; fecal coliforms, 27%; enterococci, 27%;
C. perfringens
, 61%; F-specific coliphages, ∼40%; and enteric viruses, 31%.
Cryptosporidium
oocysts and
Giardia
cysts were detected in 70% and 80%, respectively, of reclaimed water samples. Viable
Cryptosporidium
, based on cell culture infectivity assays, was detected in 20% of the reclaimed water samples. No strong correlation was found for any indicator-pathogen combination. When data for all indicators were tested using discriminant analysis, the presence/absence patterns for
Giardia
cysts,
Cryptosporidium
oocysts, infectious
Cryptosporidium
, and infectious enteric viruses were predicted for over 71% of disinfected effluents. The failure of measurements of single indicator organism to correlate with pathogens suggests that public health is not adequately protected by simple monitoring schemes based on detection of a single indicator, particularly at the detection limits routinely employed. Monitoring a suite of indicator organisms in reclaimed effluent is more likely to be predictive of the presence of certain pathogens, and a need for additional pathogen monitoring in reclaimed water in order to protect public health is suggested by this study.
Movement and Fixation of Intestinal Microbiota after Administration of Human Feces to Germfree Mice
Authors: Kibe Ryoko , Sakamoto Mitsuo , Yokota Hiroshi , Ishikawa Hiroki , Aiba Yuji , Koga Yasuhiro , Benno Yoshimi
Published: June 2005

Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of intestinal bacteria in humans, although they have some limitations as a model. Shifts in dominant species of microbiota in HFA mice after the administration of human intestinal microbiota was revealed by 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses. Characteristic terminal restriction fragments (T-RFs) were quantified as the proportion of total peak area of all T-RFs. Only the proportion of the T-RF peak at bp 366, identified as the
Gammmaproteobacteria
group and the family
Coriobacteriaceae
, was reduced in this study. Increased T-RFs over time at bp 56, 184, and 196 were affiliated with the
Clostridium
group. However, most of the isolated bacteria with unique population shifts were phylotypes. The vertical transmission of the intestinal microbiota of the mouse offspring was also investigated by dendrogram analysis derived from the similarity of T-RFLP patterns among samples. As a result, the intestinal microbiota of HFA mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications. Moreover, we revealed that human-derived lactobacilli (HDL), which have been considered difficult to colonize in the HFA mouse intestine in previous studies based on culture methods, could be detected in the HFA mouse intestine by using a lactic acid bacterium-specific primer and HDL-specific primers. Our results indicate that the intestinal microbiota of HFA mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria.
Microplate Subtractive Hybridization To Enrich for
Bacteroidales
Genetic Markers for Fecal Source Identification
Authors: Dick Linda K. , Simonich Michael T. , Field Katharine G.
Published: June 2005

The ability to identify sources of fecal pollution plays a key role in the analysis of human health risk and the implementation of water resource management strategies. One approach to this problem involves the identification of bacterial lineages or gene sequences that are found exclusively in a particular host species or group. We used subtractive hybridization to enrich for target host-specific fecal
Bacteroidales
rRNA gene fragments that were different from those of very closely related reference (subtracter) host sources. Target host rRNA gene fragments were hybridized to subtracter rRNA gene fragments immobilized in a microplate well, and target sequences that did not hybridize were cloned and sequenced for PCR primer design. The use of microplates for DNA immobilization resulted in a one-step subtractive hybridization in which the products could be directly amplified with PCR. The new host-specific primers designed from subtracted target fragments differentiated among very closely related
Bacteroidales
rRNA gene sequences and distinguished between similar fecal sources, such as elk and cow or human and domestic pet (dog).
Characterization of
Wolbachia
Transfection Efficiency by Using Microinjection of Embryonic Cytoplasm and Embryo Homogenate
Authors: Xi Zhiyong , Dobson Stephen L.
Published: June 2005

Wolbachia
spp. are intracellular alpha proteobacteria closely related to
Rickettsia
. The maternally inherited infections occur in a wide range of invertebrates, causing several reproductive abnormalities, including cytoplasmic incompatibility. The artificial transfer of
Wolbachia
between hosts (transfection) is used both for basic research examining the
Wolbachia
-host interaction and for applied strategies that use
Wolbachia
infections to affect harmful insect populations. Commonly employed transfection techniques use embryonic microinjection to transfer
Wolbachia
-infected embryo cytoplasm or embryo homogenate. Although microinjections of both embryonic cytoplasm and homogenate have been used successfully, their respective transfection efficiencies (rates of establishing stable germ line infections) have not been directly compared. Transfection efficiency may be affected by variation in
Wolbachia
quantity or quality within the donor embryos and/or the buffer types used in embryo homogenization. Here we have compared
Wolbachia
bacteria that originate from different embryonic regions for their competencies in establishing stable germ line infections. The following three buffers were compared for their abilities to maintain an appropriate in vitro environment for
Wolbachia
during homogenization and injection: phosphate-buffered saline,
Drosophila
Ringer's buffer, and a sucrose-phosphate-glutamate solution (SPG buffer). The results demonstrate that
Wolbachia
bacteria from both anterior and posterior embryo cytoplasms are competent for establishing infection, although differing survivorships of injected hosts were observed. Buffer comparison shows that embryos homogenized in SPG buffer yielded the highest transfection success. No difference was observed in transfection efficiencies when the posterior cytoplasm transfer and SPG-homogenized embryo techniques were compared. We discuss the results in relation to intra- and interspecific
Wolbachia
transfection and the future adaptation of the microinjection technique for additional insects.
Microbial Diversity in Ultra-High-Pressure Rocks and Fluids from the Chinese Continental Scientific Drilling Project in China
Authors: Zhang Gengxin , Dong Hailiang , Xu Zhiqin , Zhao Donggao , Zhang Chuanlun
Published: June 2005

Microbial communities in ultra-high-pressure (UHP) rocks and drilling fluids from the Chinese Continental Scientific Drilling Project were characterized. The rocks had a porosity of 1 to 3.5% and a permeability of ∼0.5 mDarcy. Abundant fluid and gas inclusions were present in the minerals. The rocks contained significant amounts of Fe2O3, FeO, P2O5, and nitrate (3 to 16 ppm). Acridine orange direct counting and phospholipid fatty acid analysis indicated that the total counts in the rocks and the fluids were 5.2 × 103to 2.4 × 104cells/g and 3.5 × 108to 4.2 × 109cells/g, respectively. Enrichment assays resulted in successful growth of thermophilic and alkaliphilic bacteria from the fluids, and some of these bacteria reduced Fe(III) to magnetite. 16S rRNA gene analyses indicated that the rocks were dominated by sequences similar to sequences ofProteobacteriaand that most organisms were related to nitrate reducers from a saline, alkaline, cold habitat; however, some phylotypes were either members of a novel lineage or closely related to uncultured clones. The bacterial communities in the fluids were more diverse and includedProteobacteria,Bacteroidetes, gram-positive bacteria,Planctomycetes, andCandidatustaxa. The archaeal diversity was lower, and most sequences were not related to any known cultivated species. Some archaeal sequences were 90 to 95% similar to sequences recovered from ocean sediments or other subsurface environments. Some archaeal sequences from the drilling fluids were >93% similar to sequences ofSulfolobus solfataricus, and the thermophilic nature was consistent with the in situ temperature. We inferred that the microbes in the UHP rocks reside in fluid and gas inclusions, whereas those in the drilling fluids may be derived from subsurface fluids.