bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in
Escherichia coli
by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling
Authors: Choi Jong Hyun , Lee Sang Jun , Lee Seok Jae , Lee Sang Yup 
Published: August 2003

ABSTRACT

The transcriptome profiles of recombinant
Escherichia coli
producing human insulin-like growth factor I fusion protein (IGF-I
f
) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-I
f
after induction. Among these down-regulated genes, we rationally selected and coexpressed in
E
.
coli
producing IGF-I
f
the
prsA
gene (encoding a phosphoribosyl pyrophosphate synthetase) and the
glpF
gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-I
f
could be increased from 1.8 ± 0.13 (± standard deviation) to 4.3 ± 0.24 g/liter. The volumetric productivity was also increased from 0.36 ± 0.027 to 0.82 ± 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.
An In Vitro Study of the Probiotic Potential of a Bile-Salt-Hydrolyzing
Lactobacillus fermentum
Strain, and Determination of Its Cholesterol-Lowering Properties
Authors: Pereira Dora I. A. , McCartney Anne L. , Gibson Glenn R. 
Published: August 2003

ABSTRACT

This study evaluated the use of a bile-salt-hydrolyzing
Lactobacillus fermentum
strain as a probiotic with potential hypocholesterolemic properties. The effect of
L. fermentum
on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the
Lactobacillus
strain was evaluated. Administration of
L. fermentum
resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial,
Lactobacillus
,
Bacteroides
, and clostridial populations through
L. fermentum
supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the
L. fermentum
strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that
L. fermentum
KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.
Characterization of
Streptococcus mutans
Strains Deficient in EIIAB
Man
of the Sugar Phosphotransferase System
Authors: Abranches Jacqueline , Chen Yi-Ywan M. , Burne Robert A. 
Published: August 2003

ABSTRACT

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB
Man
(encoded by
manL
) in gene regulation and sugar transport was investigated in
Streptococcus mutans
UA159. The
manL
knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB
Man
is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (
fruI
) and constitutive (
fruCD
) EII fructose, revealed that
S. mutans
EIIAB
Man
transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB
Man
controlled expression of the known virulence genes, glucosyltransferases (
gtfBC
) and fructosyltransferase (
ftf
) promoter fusions of these genes were established in the wild-type and EIIAB
Man
-deficient strains. In the
manL
mutant, the level of chloramphenicol acetyltransferase activity expressed from the
gtfBC
promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB
Man
is required for optimal expression of
gtfBC
. No significant differences were observed between the mutant and the wild-type background in
ftf
regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in
ftf
expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB
Man
-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB
Man
has a pleiotropic effect on gene expression.
Effects of a Chlorhexidine Gluconate-Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems
Authors: McBain Andrew J. , Bartolo Robert G. , Catrenich Carl E. , Charbonneau Duane , Ledder Ruth G. , Gilbert Peter 
Published: August 2003

ABSTRACT

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (
P
< 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in
Prevotella
sp. and
Selenomonas infelix
. Pure-culture studies of 10 oral bacteria (eight genera) showed that
Actinomyces naeslundii
,
Veillonella dispar
,
Prevotella nigrescens
, and the streptococci were highly susceptible to CHX, while
Lactobacillus rhamnosus
,
Fusobacterium nucleatum
, and
Neisseria subflava
were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (
P
< 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.
Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation
Authors: Higgins Vincent J. , Beckhouse Anthony G. , Oliver Anthony D. , Rogers Peter J. , Dawes Ian W. 
Published: August 2003

ABSTRACTGenome-wide expression analysis of an industrial strain ofSaccharomyces cerevisiaeduring the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of theERGgenes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similarERG10andTRR1gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed thatergmutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.
Detection and Quantification ofPlectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting
Authors: Atkins S. D. , Clark I. M. , Sosnowska D. , Hirsch P. R. , Kerry B. R. 
Published: August 2003

ABSTRACTPotato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungusPlectosphaerella cucumerinahas demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitorP. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence ofP. cucumerinain soil, root, and nematode samples. PCR was combined with a bait method to identifyP. cucumerinafrom infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A secondP.cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance ofP. cucumerinafrom two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity ofP. cucumerinato be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.
CynD, the Cyanide Dihydratase from
Bacillus pumilus
: Gene Cloning and Structural Studies
Authors: Jandhyala Dakshina , Berman Mark , Meyers Paul R. , Sewell B. Trevor , Willson Richard C. , Benedik Michael J. 
Published: August 2003

ABSTRACT

The cyanide dihydratase in
Bacillus pumilus
was shown to be an 18-subunit spiral structure by three-dimensional reconstruction of electron micrographs of negatively stained material at its optimum pH, 8.0. At pH 5.4, the subunits rearrange to form an extended left-handed helix. Gel electrophoresis of glutaraldehyde cross-linked enzyme suggests that the fundamental component of the spiral is a dimer of the 37-kDa subunit. The gene was cloned, and the recombinant enzyme was readily expressed at high levels in
Escherichia coli
. Purification of the recombinant enzyme was facilitated by the addition of a C-terminal six-histidine affinity purification tag. The tagged recombinant enzyme has

K
m

and
V
max
values similar to those published for the native enzyme. This is the first cyanide dihydratase from a gram-positive bacterium to be sequenced, and it is the first description of the structure of any member of this enzyme class. The putative amino acid sequence shares over 80% identity to the only other sequenced cyanide dihydratase, that of the gram-negative
Pseudomonas stutzeri
strain AK61, and is similar to a number of other bacterial and fungal nitrilases. This sequence similarity suggests that the novel short spiral structure may be typical of these enzymes. In addition, an active cyanide dihydratase from a non-cyanide-degrading isolate of
B
.
pumilus
(strain 8A3) was cloned and expressed. This suggests that
cynD
, the gene coding for the cyanide dihydratase, is not unique to the C1 strain of
B
.
pumilus
and is not a reflection of its origin at a mining waste site.
Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate
Cyclospora
Species from
Eimeria
Species by Multiplex PCR
Authors: Orlandi Palmer A. , Carter Laurenda , Brinker Anna Marie , da Silva Alexandre J. , Chu Dan-My , Lampel Keith A. , Monday Steven R. 
Published: August 2003

ABSTRACT

Cyclospora cayetanensis
is a coccidian parasite that causes protracted diarrheal illness in humans.
C. cayetanensis
is the only species of this genus thus far associated with human illness, although
Cyclospora
species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from
C. cayetanensis
and
Eimeria
species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between
C. cayetanensis
and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3′ end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between
C. cayetanensis
, nonhuman primate species of
Cyclospora
, and
Eimeria
species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.
N
-Acetyl-
l
-Cysteine Affects Growth, Extracellular Polysaccharide Production, and Bacterial Biofilm Formation on Solid Surfaces
Authors: Olofsson Ann-Cathrin , Hermansson Malte , Elwing Hans 
Published: August 2003

ABSTRACT

N
-Acetyl-
l
-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces.
Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism
Authors: Engebretson Jeff J. , Moyer Craig L. 
Published: August 2003

ABSTRACT

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (
n
= 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined,
Bst
UI,
Dde
I,
Sau
96I, and
Msp
I had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ≤70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).
Alteration of Chain Length Substrate Specificity of
Aeromonas caviae R
-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis
Authors: Tsuge Takeharu , Hisano Tamao , Taguchi Seiichi , Doi Yoshiharu 
Published: August 2003

ABSTRACT

Aeromonas caviae R
-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ
Ac
) is capable of providing (
R
)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ
Ac
by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ
Ac
revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using
trans
-2-octenoyl-CoA (C
8
) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C
12
) was examined by using the mutated PhaJ
Ac
as a monomer supplier in recombinant
Escherichia coli
LS5218 harboring a PHA synthase gene from
Pseudomonas
sp. strain 61-3 (
phaC1
Ps
). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C
8
) and 3-hydroxydecanoate (C
10
) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.
The Biphenyl- and 4-Chlorobiphenyl-Catabolic Transposon Tn4371, a Member of a New Family of Genomic Islands Related to IncP and Ti Plasmids
Authors: Toussaint Ariane , Merlin Christophe , Monchy Sébastien , Benotmane M. Abderrafi , Leplae Raphaël , Mergeay Max , Springael Dirk 
Published: August 2003

ABSTRACTThe nucleotide sequence of the biphenyl catabolic transposon Tn4371has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those ofAchromobacter georgiopolitanumKKS102, Tn4371carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome ofRalstonia solanacearumand on the yet-to-be-determined genomic sequences ofErwinia chrysanthemiandAzotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bphgenes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome ofRalstonia metalliduransCH34. Tn4371thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.
Dual Labeling of
Pseudomonas putida
with Fluorescent Proteins for In Situ Monitoring of Conjugal Transfer of the TOL Plasmid
Authors: Nancharaiah Y. Venkata , Wattiau Pierre , Wuertz Stefan , Bathe Stephan , Mohan S. Venkata , Wilderer Peter A. , Hausner Martina 
Published: August 2003

ABSTRACT

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to
Pseudomonas putida
KT2442, which was chromosomally labeled with
dsRed
by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor
P. putida
cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.