bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic Conversion of Averufin to Hydroxyversicolorone and Elucidation of a Novel Metabolic Grid Involved in Aflatoxin Biosynthesis
Authors: Yabe Kimiko , Chihaya Naomi , Hamamatsu Shioka , Sakuno Emi , Hamasaki Takashi , Nakajima Hiromitsu , Bennett J. W.
Published: January 2003

The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from
Aspergillus parasiticus
. When (1′
S
,5′
S
)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1′
R
,5′
R
)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with
A. parasiticus
NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1′
S
,5′
S
)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.
A Colonization Factor (Production of Lateral Flagella) of Mesophilic
Aeromonas
spp. Is Inactive in
Aeromonas salmonicida
Strains
Authors: Merino Susana , Gavín Rosalina , Vilches Silvia , Shaw Jonathan G. , Tomás Juan M.
Published: January 2003

The nine
laf
(lateral flagellum) genes of mesophilic aeromonads are in the
Aeromonas salmonicida
genome. The
laf
genes are functional, except for
lafA
(flagellin gene), which was inactivated by transposase 8 (IS
3
family). A pathogenic characteristic of mesophilic aeromonads (lateral flagella) is abolished in this specialized pathogen with a narrow host range.
Inactivation of
Salmonella enterica
Serovar Enteritidis by Ultrasonic Waves under Pressure at Different Water Activities
Authors: Álvarez I. , Mañas P. , Sala F. J. , Condón S.
Published: January 2003

The inactivation of
Salmonella enterica
serovar Enteritidis by ultrasonic waves (20 kHz; 117-μm wavelength) under pressure (175 kPa) at nonlethal temperatures (manosonication [MS]) and lethal temperatures (manothermosonication [MTS]) in media of different water activities has been investigated. Heat decimal reduction time values increased 30 times when the water activity was decreased from nearly 1 to 0.96, but the MS resistance was increased only twofold. The inactivation of
Salmonella
serovar Enteritidis by ultrasound under pressure at low water activities was a phenomenon of the “all-or-nothing” type. A synergistic lethal effect was observed between heat and ultrasound in media with reduced water activity; the lower the water activity, the greater the synergistic effect. This work could be useful for improving sanitation and preservation treatments of foods, especially those which are sensitive to temperature and those in which components protect microorganisms to heat. It also contributes to our knowledge of microbial inactivation mechanisms by MS and MTS treatments.
Degradation and Transformability of DNA from Transgenic Leaves
Authors: Ceccherini MariaTeresa , Poté John , Kay Elisabeth , Van Van Tran , Maréchal Joëlle , Pietramellara Giacomo , Nannipieri Paolo , Vogel Timothy M. , Simonet Pascal
Published: January 2003

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by
Erwinia chrysanthemi
, and attack by the plant pathogen
Ralstonia solanacearum
. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable
aadA
sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.
Molecular Characterization of Natural
Erwinia pyrifoliae
Strains Deficient in Hypersensitive Response
Authors: Jock Susanne , Kim Won-Sik , Barny Marie-Anne , Geider Klaus
Published: January 2003

From necrotic tissue of a Nashi pear tree, 24
Erwinia pyrifoliae
strains, found to be identical by pulsed-field gel electrophoresis analysis, were isolated. Thirteen strains were not virulent on immature pears and did not induce a hypersensitive response in tobacco leaves. The defective gene
hrpL
was complemented with intact genes from
E. pyrifoliae
and
Erwinia amylovora
.
Comparison of UV Inactivation of Spores of Three
Encephalitozoon
Species with That of Spores of Two DNA Repair-Deficient
Bacillus subtilis
Biodosimetry Strains
Authors: Marshall Marilyn M. , Hayes Samuel , Moffett Jackie , Sterling Charles R. , Nicholson Wayne L.
Published: January 2003

When exposed to 254-nm UV, spores of
Encephalitozoon intestinalis
,
Encephalitozoon cuniculi
, and
Encephalitozoon hellem
exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m
2
, respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant
Bacillus subtilis
strains used as biodosimetry surrogates. The results indicate that spores of
Encephalitozoon
spp. are readily inactivated at low UV fluences and that spores of UV-sensitive
B. subtilis
strains can be useful surrogates in evaluating UV reactor performance.
Effect of Stress on the Ability of a
phlA
-Based Quantitative Competitive PCR Assay To Monitor Biocontrol Strain
Pseudomonas fluorescens
CHA0
Authors: Rezzonico Fabio , Moënne-Loccoz Yvan , Défago Geneviève
Published: January 2003

A quantitative competitive PCR (QC-PCR) assay targeting the
phlA
gene of
Pseudomonas fluorescens
CHA0 was developed and tested in vitro. Statistically significant, positive correlations were found between QC-PCR and both CFU and total cell number when studying cells in log or stationary phase. The correlations disappeared when considering stressed cells.
Characteristics of a Novel Type of Bovine
Cryptosporidium andersoni
Authors: Satoh Masaaki , Hikosaka Kenji , Sasaki Takako , Suyama Yoshihisa , Yanai Tokuma , Ohta Minoru , Nakai Yutaka
Published: January 2003

We isolated oocysts that resemble
Cryptosporidium andersoni
from cattle grazing on a farm in Japan. The partial sequences of genes from the isolate were coincident with published sequences of genes of
C. andersoni
. Since the isolate was able to infect SCID mice, the isolate appears to be a novel type of
C. andersoni
.
Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups
Authors: Leaphart A. B. , Friez M. J. , Lovell C. R.
Published: January 2003

Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including
Spartina alterniflora
,
Salicornia virginica
, and
Juncus roemerianus
. Only
S. alterniflora
roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with
Sphingomonas paucimobilis ligH
were also recovered.
Molecular Typing of
Aeromonas
Isolates in Natural Mineral Waters
Authors: Villari P. , Crispino M. , Montuori P. , Boccia S.
Published: January 2003

A total of 103 isolates of
Aeromonas
spp. were obtained over a 3-year period from a natural mineral water and from surface streams located within the boundaries of the watershed of the natural mineral water wells and were typed by macrorestriction analysis of genomic DNA with
Xba
I and by pulsed-field gel electrophoresis. All
Aeromonas caviae
isolates from the natural mineral water belonged to the same clone, and an analogous clonal identity was found among
Aeromonas hydrophila
isolates. These two clones expressed no hemolytic or cytotoxic activities.
Aeromonas
isolates from surface waters showed high molecular heterogeneity and were not related to the clones found in the natural mineral water. The presence of aeromonads chronically found in the natural mineral water was a likely consequence of a localized development of a biofilm, with no exogenous contamination of the aquifer. Molecular fingerprinting of drinking water isolates is a useful tool in explaining possible reasons for bacterial occurrences.
A Food-Grade Approach for Functional Analysis and Modification of Native Plasmids in
Lactococcus lactis
Authors: Cotter Paul D. , Hill Colin , Ross R. Paul
Published: January 2003

While plasmids from lactic acid bacteria possess many traits that are of industrial value, their exploitation is often frustrated by an inability to conduct food-grade engineering of native plasmids or to readily screen for their transfer. Here we describe a system that uses a RepA
+
temperature-sensitive helper plasmid and a RepA
−
cloning vector to overcome these problems while maintaining the food-grade status of the native plasmid. This strategy was used to precisely delete
ltnA1
alone, or in conjunction with
ltnA2
(encoding the structural proteins of the lantibiotic lacticin 3147), from the native 60.2-kb plasmid pMRC01 and to select for the transfer of pMRC01 between
Lactococcus lactis
strains.
Glutathione
S
-Transferase Isoenzymes from
Streptomyces griseus
Authors: Dhar Kajari , Dhar Alok , Rosazza John P. N.
Published: January 2003

An inducible, cytosolic glutathione
S
-transferase (GST) was purified from
Streptomyces griseus
. GST isoenzymes with pI values of 6.8 and 7.9 used standard GST substrates including 1-chloro-2,4-dinitrobenzene. GST had subunit and native
M
r
s of 24 and 48, respectively, and the N-terminal sequence SMILXYWDIIRGLPAH.
Survival and Resuscitation of Ten Strains of
Campylobacter jejuni
and
Campylobacter coli
under Acid Conditions
Authors: Chaveerach P. , ter Huurne A. A. H. M. , Lipman L. J. A. , van Knapen F.
Published: January 2003

The culturability of 10 strains of
Campylobacter jejuni
and
Campylobacter coli
was studied after the bacteria were exposed to acid conditions for various periods of time.
Campylobacter
cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10
Campylobacter
strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4′,6′-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of
Campylobacter
could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.
Disruption of the
CAR1
Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast
Saccharomyces cerevisiae
Authors: Shima Jun , Sakata-Tsuda Yuko , Suzuki Yasuo , Nakajima Ryouichi , Watanabe Hajime , Kawamoto Shinichi , Takano Hiroyuki
Published: January 2003

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the
CAR1
gene enhances freeze tolerance.
Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat
Authors: Horn Marcus A. , Matthies Carola , Küsel Kirsten , Schramm Andreas , Drake Harold L.
Published: January 2003

The emission of methane (1.3 mmol of CH
4
m
−2
day
−1
), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH
4
(0.4 to 1.7 μmol g [dry wt] of soil
−1
day
−1
) under anoxic conditions. At in situ pH, supplemental H
2
-CO
2
, ethanol, and 1-propanol all increased CH
4
production rates while formate, acetate, propionate, and butyrate inhibited the production of CH
4
; methanol had no effect. H
2
-dependent acetogenesis occurred in H
2
-CO
2
-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H
2
that were subsequently consumed. The accumulation of H
2
was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H
2
; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H
2
-utilizing methanogens. A total of 10
9
anaerobes and 10
7
hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H
2
-CO
2
-supplemented, fatty acid-enriched defined medium. CH
4
production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of
Methanobacteriaceae
,
Methanomicrobiales
, and
Methanosarcinaceae
were retrieved from the highest CH
4
-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H
2
that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H
2
is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the
Methanobacteriaceae
,
Methanomicrobiales
, and
Methanosarcinaceae
.
Intergeneric Conjugation in
Streptomyces peucetius
and
Streptomyces
sp
.
Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the
chiC
Gene
Authors: Paranthaman Senthamaraikannan , Dharmalingam Kuppamuthu
Published: January 2003

Intergeneric conjugal transfer of plasmid DNA from
Escherichia coli
to S
treptomyces
circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of
Streptomyces
protoplasts. The anthracycline antibiotic-producing strains
Streptomyces peucetius
and
Streptomyces
sp. strain C5 were transformed using
E. coli
ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with
Streptomyces
strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (
attB
) of the
Streptomyces
genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and
S. peucetius
, is TTC. These results also showed that the φC31 integrative recombination is active and the phage
attP
site is functional in
S. peucetius
as well as in C5. The efficiency and specificity of φC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant
chiC
into the wild-type
S. peucetius
chromosome led to the production of 30-fold more chitinase.
Enterococci as Indicators of Lake Michigan Recreational Water Quality: Comparison of Two Methodologies and Their Impacts on Public Health Regulatory Events
Authors: Kinzelman Julie , Ng Clement , Jackson Emma , Gradus Stephen , Bagley Robert
Published: January 2003

The frequency of poor-water-quality advisories issued in Milwaukee and Racine, Wisconsin, in the absence of identifiable sources of contamination brought into question the reliability of the present indicator organism,
Escherichia coli
. Enteroccoci have been suggested as an alternative to
E. coli
for freshwater monitoring due to their direct correlation to swimmer-associated gastroenteritis. The purpose of this research was threefold: (i) to explore enterococci as an alternative to
E. coli
for monitoring freshwater Lake Michigan beaches, (ii) to evaluate the impact of the two indicators on regulatory decisions, and (iii) to compare membrane filtration m-enterococcus agar with indoxyl-β-
d
-glucoside to a chemical substrate technique (Enterolert) for the recovery of enterococci. Recreational water samples from Milwaukee (
n
= 305) and Racine (
n
= 153) were analyzed for the enumeration of
E. coli
and enterococci using IDEXX Colilert-18 and Enterolert. Correlation between the indicators was low (
R
2
= 0.60 and 0.69). Based on U.S. Environmental Protection Agency bacterial indicator threshold levels of risk for full body immersion, using enterococci would have resulted in 56 additional unsafe-recreational-water-quality advisories compared to the total from using
E. coli
and the substrate-based methods. A comparison of the two enterococcal methods (
n
= 124) yielded similar results (
R
2
= 0.62). This was further confounded by the frequent inability to verify enterococci from those wells producing fluorescence by the defined substrate test using conventional microbiological methods. These results suggest that further research is necessary regarding the use of defined substrate technology interchangeably with the U.S. Environmental Protection Agency-approved membrane filtration test for the detection of enterococci from fresh surface water.
Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation
Authors: Burton Jeremy P. , Cadieux Peter A. , Reid Gregor
Published: January 2003

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to
Lactobacillus
species sequences. Sequences homologous to
Lactobacillus iners
sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.
Comparison of Serological Assays for Detection of
Chlamydia trachomatis
Antibodies in Different Groups of Obstetrical and Gynecological Patients
Authors: Bax C. J. , Mutsaers J. A. E. M. , Jansen C. L. , Trimbos J. B. , Dörr P. J. , Oostvogel P. M.
Published: January 2003

New serological enzyme immunoassays (EIAs) were compared with microimmunofluorescence (MIF) as a “gold standard” to detect
Chlamydia trachomatis
antibodies in different groups of obstetrical, gynecological, and subfertile patients. There were no significant differences in seroprevalence rates, except for the group of
C. trachomatis
-positive patients (
P
< 0.01). Test characteristics were calculated for Chlamydia-EIA (Biologische Analysensystem GmbH, Lich, Germany) and pELISA (Medac, Wedel, Germany). pELISA seems to be a good alternative to MIF. It has high specificity and is easier to perform.
Specific Detection of Human BK Polyomavirus in Urine Samples of Immunocompromised Patients
Authors: Holman Carol J. , van Burik Jo-Anne H. , Hinrichs Steven H. , Balfour Henry H.
Published: January 2003

A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were postive when specimens were submitted because of clinical suspicion of BK virus infection.
The Scientist's World
Authors: Davis Bernard D.
Published: January 2003

This paper describes the features of the world of science, and it compares that world briefly with that of politics and the law. It also discusses some “postmodern” trends in philosophy and sociology that have been undermining confidence in the objectivity of science and thus have contributed indirectly to public mistrust. The paper includes broader implications of interactions of government and science.
Genetics of Mosquito Vector Competence
Authors: Beerntsen Brenda T. , James Anthony A. , Christensen Bruce M.
Published: January 2003

Mosquito-borne diseases are responsible for significant human morbidity and mortality throughout the world. Efforts to control mosquito-borne diseases have been impeded, in part, by the development of drug-resistant parasites, insecticide-resistant mosquitoes, and environmental concerns over the application of insecticides. Therefore, there is a need to develop novel disease control strategies that can complement or replace existing control methods. One such strategy is to generate pathogen-resistant mosquitoes from those that are susceptible. To this end, efforts have focused on isolating and characterizing genes that influence mosquito vector competence. It has been known for over 70 years that there is a genetic basis for the susceptibility of mosquitoes to parasites, but until the advent of powerful molecular biological tools and protocols, it was difficult to assess the interactions of pathogens with their host tissues within the mosquito at a molecular level. Moreover, it has been only recently that the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production, have been investigated. The molecular characterization of genes that influence vector competence is becoming routine, and with the development of the Sindbis virus transducing system, potential antipathogen genes now can be introduced into the mosquito and their effect on parasite development can be assessed in vivo. With the recent successes in the field of mosquito germ line transformation, it seems likely that the generation of a pathogen-resistant mosquito population from a susceptible population soon will become a reality.
Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals
Authors: van Geest Marleen , Lolkema Juke S.
Published: January 2003

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.
Higher Levels of Organization in the Interphase Nucleus of Cycling and Differentiated Cells
Authors: Leitch Andrew R.
Published: January 2003

The review examines the structured organization of interphase nuclei using a range of examples from the plants, animals, and fungi. Nuclear organization is shown to be an important phenomenon in cell differentiation and development. The review commences by examining nuclei in dividing cells and shows that the organization patterns can be dynamic within the time frame of the cell cycle. When cells stop dividing, derived differentiated cells often show quite different nuclear organizations. The developmental fate of nuclei is divided into three categories. (i) The first includes nuclei that undergo one of several forms of polyploidy and can themselves change in structure during the course of development. Possible function roles of polyploidy is given. (ii) The second is nuclear reorganization without polyploidy, where nuclei reorganize their structure to form novel arrangements of proteins and chromosomes. (iii) The third is nuclear disintegration linked to programmed cell death. The role of the nucleus in this process is described. The review demonstrates that recent methods to probe nuclei for nucleic acids and proteins, as well as to examine their intranuclear distribution in vivo, has revealed much about nuclear structure. It is clear that nuclear organization can influence or be influenced by cell activity and development. However, the full functional role of many of the observed phenomena has still to be fully realized.
Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily
Authors: Dunwell Jim M. , Khuri Sawsan , Gane Paul J.
Published: January 2003

This review summarizes the recent discovery of the cupin superfamily (from the Latin term “cupa,” a small barrel) of functionally diverse proteins that initially were limited to several higher plant proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins, and auxin-binding protein. Knowledge of the three-dimensional structure of two vicilins, seed proteins with a characteristic β-barrel core, led to the identification of a small number of conserved residues and thence to the discovery of several microbial proteins which share these key amino acids. In particular, there is a highly conserved pattern of two histidine-containing motifs with a varied intermotif spacing. This cupin signature is found as a central component of many microbial proteins including certain types of phosphomannose isomerase, polyketide synthase, epimerase, and dioxygenase. In addition, the signature has been identified within the N-terminal effector domain in a subgroup of bacterial AraC transcription factors. As well as these single-domain cupins, this survey has identified other classes of two-domain bicupins including bacterial gentisate 1,2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases, fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupin evolution is discussed from the perspective of the structure-function relationships, using data from the genomes of several prokaryotes, especially Bacillus subtilis. Many of these functions involve aspects of sugar metabolism and cell wall synthesis and are concerned with responses to abiotic stress such as heat, desiccation, or starvation. Particular emphasis is also given to the oxalate-degrading enzymes from microbes, their biological significance, and their value in a range of medical and other applications.
Molecular Basis of Symbiotic Promiscuity
Authors: Perret Xavier , Staehelin Christian , Broughton William J.
Published: January 2003

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the “keys” to a succession of legume “doors”. Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.
Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process
Authors: Woese Carl R. , Olsen Gary J. , Ibba Michael , Söll Dieter
Published: January 2003

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the “gemini group.” (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.
Metabolic Engineering of
Saccharomyces cerevisiae
Authors: Ostergaard Simon , Olsson Lisbeth , Nielsen Jens
Published: January 2003

Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.
Hepatitis B Virus Biology
Authors: Seeger Christoph , Mason William S.
Published: January 2003

Hepadnaviruses (hepatitis B viruses) cause transient and chronic infections of the liver. Transient infections run a course of several months, and chronic infections are often lifelong. Chronic infections can lead to liver failure with cirrhosis and hepatocellular carcinoma. The replication strategy of these viruses has been described in great detail, but virus-host interactions leading to acute and chronic disease are still poorly understood. Studies on how the virus evades the immune response to cause prolonged transient infections with high-titer viremia and lifelong infections with an ongoing inflammation of the liver are still at an early stage, and the role of the virus in liver cancer is still elusive. The state of knowledge in this very active field is therefore reviewed with an emphasis on past accomplishments as well as goals for the future.
Virioplankton: Viruses in Aquatic Ecosystems
Authors: Wommack K. Eric , Colwell Rita R.
Published: January 2003

The discovery that viruses may be the most abundant organisms in natural waters, surpassing the number of bacteria by an order of magnitude, has inspired a resurgence of interest in viruses in the aquatic environment. Surprisingly little was known of the interaction of viruses and their hosts in nature. In the decade since the reports of extraordinarily large virus populations were published, enumeration of viruses in aquatic environments has demonstrated that the virioplankton are dynamic components of the plankton, changing dramatically in number with geographical location and season. The evidence to date suggests that virioplankton communities are composed principally of bacteriophages and, to a lesser extent, eukaryotic algal viruses. The influence of viral infection and lysis on bacterial and phytoplankton host communities was measurable after new methods were developed and prior knowledge of bacteriophage biology was incorporated into concepts of parasite and host community interactions. The new methods have yielded data showing that viral infection can have a significant impact on bacteria and unicellular algae populations and supporting the hypothesis that viruses play a significant role in microbial food webs. Besides predation limiting bacteria and phytoplankton populations, the specific nature of virus-host interaction raises the intriguing possibility that viral infection influences the structure and diversity of aquatic microbial communities. Novel applications of molecular genetic techniques have provided good evidence that viral infection can significantly influence the composition and diversity of aquatic microbial communities.
Translational Control of Viral Gene Expression in Eukaryotes
Authors: Gale Michael , Tan Seng-Lai , Katze Michael G.
Published: January 2003

As obligate intracellular parasites, viruses rely exclusively on the translational machinery of the host cell for the synthesis of viral proteins. This relationship has imposed numerous challenges on both the infecting virus and the host cell. Importantly, viruses must compete with the endogenous transcripts of the host cell for the translation of viral mRNA. Eukaryotic viruses have thus evolved diverse mechanisms to ensure translational efficiency of viral mRNA above and beyond that of cellular mRNA. Mechanisms that facilitate the efficient and selective translation of viral mRNA may be inherent in the structure of the viral nucleic acid itself and can involve the recruitment and/or modification of specific host factors. These processes serve to redirect the translation apparatus to favor viral transcripts, and they often come at the expense of the host cell. Accordingly, eukaryotic cells have developed antiviral countermeasures to target the translational machinery and disrupt protein synthesis during the course of virus infection. Not to be outdone, many viruses have answered these countermeasures with their own mechanisms to disrupt cellular antiviral pathways, thereby ensuring the uncompromised translation of virion proteins. Here we review the varied and complex translational programs employed by eukaryotic viruses. We discuss how these translational strategies have been incorporated into the virus life cycle and examine how such programming contributes to the pathogenesis of the host cell.
Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast
Authors: Contamine Véronique , Picard Marguerite
Published: January 2003

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho
+
mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho
0
petites) and/or lead to truncated forms (rho
−
) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho
+
genome depends on a centromere-like structure dispensable for the maintenance of rho
−
mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.
Life History and Developmental Processes in the BasidiomyceteCoprinus cinereus
Authors: Kües Ursula
Published: January 2003

Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, β-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.
A Functional-Phylogenetic Classification System for Transmembrane Solute Transporters
Authors: Saier Milton H.
Published: January 2003

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects.
Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)
Authors: Murray Noreen E.
Published: January 2003

Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.