bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distribution of Environmental Mycobacteria in Karonga District, Northern Malawi
Authors: Chilima Benson Z. , Clark Ian M. , Floyd Sian , Fine Paul E. M. , Hirsch Penny R. 
Published: April 2006

ABSTRACTThe genusMycobacteriumincludes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis,Mycobacterium bovisBCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains ofMycobacterium fortuitum.This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.
Quantitative Real-Time PCR Analysis of FecalLactobacillusSpecies in Infants Receiving a Prebiotic Infant Formula
Authors: Haarman Monique , Knol Jan 
Published: April 2006

ABSTRACTThe developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally,BifidobacteriumandLactobacilluspredominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, theLactobacillusspecies distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5′ nuclease assays, targeted on rRNA intergenic spacer regions, were developed forLactobacillus acidophilus,Lactobacillus casei,Lactobacillus delbrueckii,Lactobacillus fermentum,Lactobacillus paracasei,Lactobacillus plantarum,Lactobacillus reuteri, andLactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of differentLactobacillusspecies in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% ± 0.3% versus 4.1% ± 1.5%) and the OSF group (0.8% ± 0.3% versus 4.4% ± 1.4%). TheLactobacillusspecies distribution in the OSF group was comparable to breast-fed infants, with relatively high levels ofL. acidophilus,L. paracasei, andL. casei. The SF-fed infants, on the other hand, contained moreL. delbrueckiiand lessL. paracaseicompared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.
Evidence for Outcrossing via the Buller Phenomenon in a Substrate Simultaneously Inoculated with Spores and Mycelium of
Agaricus bisporus
Authors: Callac Philippe , Spataro Cathy , Caille Aurélie , Imbernon Micheline 
Published: April 2006

ABSTRACT

In
Agaricus bisporus
, traditional cultivars and most of the wild populations belong to
A. bisporus
var.
bisporus
, which has a predominantly pseudohomothallic life cycle in which most meiospores are heterokaryons (
n
+
n
). A lower proportion of homokaryotic (
n
) meiospores, which typify the heterothallic life cycle, also are produced. In wild populations, pseudohomothallism was thought previously to play a major role, but recent analyses have found that significant outcrossing also may occur. We inoculated a standard substrate for
A. bisporus
cultivation simultaneously with homokaryotic mycelium from one parent and spores from a second parent. Culture trays produced numerous sporocarps that could theoretically have resulted from five different reproductive modes (pseudohomothallism, selfing or outcrossing via heterothallism, and selfing or outcrossing via the Buller phenomenon [i.e., between a homokaryon and a heterokaryon]). Most or all of the sporocarps resulted from outcrossing between the inoculated homokaryon and the inoculated heterokaryotic spores (or mycelia that grew from them). These data broaden our understanding of population dynamics under field conditions and provide an outcrossing method that could be used in commercial breeding programs.
Natural Electrotransformation of Lightning-Competent
Pseudomonas
sp. Strain N3 in Artificial Soil Microcosms
Authors: Cérémonie Hélène , Buret François , Simonet Pascal , Vogel Timothy M. 
Published: April 2006

ABSTRACT

The lightning-competent
Pseudomonas
sp. strain N3, recently isolated from soil, has been used to study the extent of natural electrotransformation (NET) or lightning transformation as a horizontal gene transfer mechanism in soil. The variation of electrical fields applied to the soil with a laboratory-scale lightning system provides an estimate of the volume of soil affected by NET. Based on the range of the electric field that induces NET of
Pseudomonas
strain N3, the volume of soil, where NET could occur, ranges from 2 to 950 m
3
per lightning strike. The influence of DNA parameters (amount, size, and purity) and DNA soil residence time were also investigated. NET frequencies (electrotransformants/recipient cells) ranged from 10
−8
for cell lysate after 1 day of residence in soil to 4 × 10
−7
with a purified plasmid added immediately before the lightning. The electrical field gradient (in kilovolts per cm) also played a role as NET frequencies ranging from 1 × 10
−5
at 2.3 kV/cm to 1.7 × 10
−4
at 6.5 kV/cm.
Reevaluation of the Life Cycle of
Tuber magnatum
Authors: Paolocci Francesco , Rubini Andrea , Riccioni Claudia , Arcioni Sergio 
Published: April 2006

ABSTRACT

Tuber
spp. are ectomycorrhizal ascomycetes that produce ascocarps known as truffles. Basic aspects of
Tuber
biology have yet to be fully elucidated. In particular, there are conflicting hypotheses concerning the mating system and the ploidy level of the mycorrhizal and truffle hyphae. We used polymorphic microsatellites to compare the allelic configurations of asci with those from the network of the surrounding hyphae in single
Tuber magnatum
truffles. We then used these truffles to inoculate host plants and evaluated the microsatellite configurations of the resulting mycorrhizal root tips. These analyses provide direct evidence that
T. magnatum
outcrosses and that its life cycle is predominantly haploid. In addition to its scientific significance, this basic understanding of the
T. magnatum
life cycle may have practical importance in developing strategies to obtain and select nursery-produced mycorrhizal plants as well as in the management of artificial plantations of this and other
Tuber
spp.
A Mono-2-Ethylhexyl Phthalate Hydrolase from a
Gordonia
sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate
Authors: Nishioka Tuguhiro , Iwata Makoto , Imaoka Takuya , Mutoh Maiko , Egashira Yoshihiro , Nishiyama Takashi , Shin Takashi , Fujii Takao 
Published: April 2006

ABSTRACT

Gordonia
sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The

K
m

and
V
max
values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of
meta
-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.
Development and Characterization of Stable Sediment-Free Anaerobic Bacterial Enrichment Cultures That Dechlorinate Aroclor 1260
Authors: Bedard Donna L. , Bailey Jessica J. , Reiss Brandon L. , Jerzak Greta Van Slyke 
Published: April 2006

ABSTRACTWe have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarilymetadechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H2. Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 μg/ml of Aroclor 1260 at 22 to 24°C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 μg/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H2. Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence ofThauera-likeBetaproteobacteria,Geobacter-likeDeltaproteobacteria,Pseudomonasspecies, variousClostridiales,Bacteroidetes,Dehalococcoidesof theChloroflexigroup, and unclassifiedEubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications.
Emergence of Distinct Genotypes of
Cryptosporidium parvum
in Structured Host Populations
Authors: Tanriverdi Sultan , Markovics Alex , Arslan M. Özkan , Itik Aysel , Shkap Varda , Widmer Giovanni 
Published: April 2006

ABSTRACT

Cryptosporidium parvum
is an apicomplexan parasite that infects humans and ruminants.
C. parvum
isolated from cattle in northeastern Turkey and in Israel was genotyped using multiple polymorphic genetic markers, and the two populations were compared to assess the effect of cattle husbandry on the parasite's population structure. Dairy herds in Israel are permanently confined with essentially no opportunity for direct herd-to-herd transmission, whereas in Turkey there are more opportunities for transmission as animals range over wider areas and are frequently traded. A total of 76
C. parvum
isolates from 16 locations in Israel and seven farms in the Kars region in northeastern Turkey were genotyped using 16 mini- and microsatellite markers. Significantly, in both countries distinct multilocus genotypes confined to individual farms were detected. The number of genotypes per farm was higher and mixed isolates were more frequent in Turkey than in Israel. As expected from the presence of distinct multilocus genotypes in individual herds, linkage disequilibrium among loci was detected in Israel. Together, these observations show that genetically distinct populations of
C. parvum
can emerge within a group of hosts in a relatively short time. This may explain the frequent detection of host-specific genotypes with unknown taxonomic status in surface water and the existence of geographically restricted
C. hominis
genotypes in humans.
An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes
Authors: Bloor Alexandra E. , Cranenburgh Rocky M. 
Published: April 2006

ABSTRACT

A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by
dif
sites and regions homologous to the chromosomal target locus.
dif
is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in
Escherichia coli
and RipX/CodV in
Bacillus subtilis
. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated
dif
sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in
trans
; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.
Cloning and Sequencing of the
ompA
Gene of
Enterobacter sakazakii
and Development of an
ompA
-Targeted PCR for Rapid Detection of
Enterobacter sakazakii
in Infant Formula
Authors: Mohan Nair Manoj Kumar , Venkitanarayanan Kumar S. 
Published: April 2006

ABSTRACT

Enterobacter sakazakii
is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify
E. sakazakii
. The outer membrane protein A gene (
ompA
), along with its flanking sequences from
E. sakazakii
(ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the
ompA
gene with other sequences available in the GenBank database revealed a high degree of homology with
ompA
genes of other gram-negative bacteria belonging to the
Enterobacteriaceae
. Based on regions of the
ompA
gene unique to
E. sakazakii
, two primers were synthesized to develop and optimize an
E. sakazakii
-specific PCR. The PCR amplified a 469-bp DNA product from all
E. sakazakii
strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10
3
CFU/ml of
E. sakazakii
bacteria in infant formula directly and 10
−1
CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of
E. sakazakii
in infant formula.
Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase
Authors: Zhang Wenjun , Ames Brian D. , Tsai Shiou-Chuan , Tang Yi 
Published: April 2006

ABSTRACT

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (
oxy
and
otc
genes) PKS genes from
Streptomyces rimosus
. Sequence analysis revealed a total of 21 genes between the
otrA
and
otrB
resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the
oxy
-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the
oxy
-encoded minimal PKS possesses novel starter unit specificity.
Toxicity and Mode of Action of
Bacillus thuringiensis
Cry Proteins in the Mediterranean Corn Borer,
Sesamia nonagrioides
(Lefebvre)
Authors: González-Cabrera Joel , Farinós Gema P. , Caccia Silvia , Díaz-Mendoza Mercedes , Castañera Pedro , Leonardi Maria Giovanna , Giordana Barbara , Ferré Juan 
Published: April 2006

ABSTRACT

Sesamia nonagrioides
is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the
Bacillus thuringiensis
Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on
S. nonagrioides
(binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with
125
I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC
3
(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to
Ostrinia nubilalis
, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.
Thiamine-Auxotrophic Mutants of
Pseudomonas fluorescens
CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression
Authors: Dubuis Christophe , Rolli Joëlle , Lutz Matthias , Défago Geneviève , Haas Dieter 
Published: April 2006

ABSTRACT

In the biocontrol strain
Pseudomonas fluorescens
CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to
N
-acyl-homoserine lactones. Transposon Tn
5
was used to isolate
P. fluorescens
CHA0 insertion mutants that expressed an
rsmZ-gfp
fusion at reduced levels. Five of these mutants were
gacS
negative, and in them the
gacS
mutation could be complemented for exoproduct and signal synthesis by the
gacS
wild-type allele. Furthermore, two thiamine-auxotrophic (
thiC
) mutants that exhibited decreased signal synthesis in the presence of 5 × 10
−8
M thiamine were found. Under these conditions, a
thiC
mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a
thiC
mutant was impaired for biological control of
Pythium ultimum
on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by
P. fluorescens
.
Very Low Ethanol Concentrations Affect the Viability and Growth Recovery in Post-Stationary-Phase
Staphylococcus aureus
Populations
Authors: Chatterjee Indranil , Somerville Greg A. , Heilmann Christine , Sahl Hans-Georg , Maurer Hans H. , Herrmann Mathias 
Published: April 2006

ABSTRACT

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (≤0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for
Staphylococcus aureus
strain DSM20231 with VLEC (VLEC
+
conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC
−
conditions) for prolonged culture times (up to 8 days). VLEC
+
-grown late-stationary-phase
S. aureus
displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of
S. aureus
post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC
+
conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC
+
conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of
S. aureus
even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of
S. aureus
is lower than that previously reported.