bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of Stress on Individual Lag Time Distributions of
Listeria monocytogenes
Authors: Guillier L. , Pardon P. , Augustin J.-C. 
Published: June 2005

ABSTRACT

The effects of nine common food industry stresses on the times to the turbidity (
T
d
) distribution of
Listeria monocytogenes
were determined. It was established that the main source of the variability of
T
d
for stressed cells was the variability of individual lag times. The distributions of
T
d
revealed that there was a noticeable difference in response to the stresses encountered by the
L. monocytogenes
cells. The applied stresses led to significant changes of the shape, the mean, and the variance of the distributions. The variance of
T
d
of wells inoculated with single cells issued from a culture in the exponential growth phase was multiplied by at least 6 and up to 355 for wells inoculated with stressed cells. These results suggest stress-induced variability may be important in determining the reliability of predictive microbiological models.
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

ABSTRACTThe 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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTLactobacillus 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.