bluuebunny/crossref_metadata_2025 huggingface dataset:
Biotransformation of 2,4,6,8,10,12-Hexanitro-2,4,6,8,10,12-Hexaazaisowurtzitane (CL-20) by Denitrifying
Pseudomonas
sp. Strain FA1
Authors: Bhushan Bharat , Paquet Louise , Spain Jim C. , Hawari Jalal
Published: September 2003

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source,
Pseudomonas
sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 ± 0.1 nmol h
−1
mg of cell biomass
−1
and 11.5 ± 0.4 nmol h
−1
mg of protein
−1
, respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO
2
−
), 1.5 molecules of nitrous oxide (N
2
O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.
PAC1
, a pH-Regulatory Gene from
Fusarium verticillioides
Authors: Flaherty Joseph E. , Pirttilä Anna Maria , Bluhm Burton H. , Woloshuk Charles P.
Published: September 2003

Fumonisins are a group of mycotoxins that contaminate maize and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice. Fumonisin biosynthesis in
Fusarium verticillioides
is repressed by nitrogen and alkaline pH. We cloned a
PACC
-like gene (
PAC1
) from
F. verticillioides
.
PACC
genes encode the major transcriptional regulators of several pH-responsive pathways in other filamentous fungi. In Northern blot analyses, a
PAC1
probe hybridized to a 2.2-kb transcript present in
F. verticillioides
grown at alkaline pH. A mutant of
F. verticillioides
with a disrupted
PAC1
gene had severely impaired growth at alkaline pH. The mutant produced more fumonisin than the wild type when grown on maize kernels and in a synthetic medium buffered at an acidic pH, 4.5. The mutant, but not the wild type, also produced fumonisin B
1
when mycelia were resuspended in medium buffered at an alkaline pH, 8.4. Transcription of
FUM1
, a gene involved in fumonisin biosynthesis, was correlated with fumonisin production. We conclude that
PAC1
is required for growth at alkaline pH and that
Pac1
may have a role as a repressor of fumonisin biosynthesis under alkaline conditions.
Expression Levels of the Yeast Alcohol Acetyltransferase GenesATF1,Lg-ATF1, andATF2Control the Formation of a Broad Range of Volatile Esters
Authors: Verstrepen Kevin J. , Van Laere Stijn D. M. , Vanderhaegen Bart M. P. , Derdelinckx Guy , Dufour Jean-Pierre , Pretorius Isak S. , Winderickx Joris , Thevelein Johan M. , Delvaux Freddy R.
Published: September 2003

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the knownSaccharomyces cerevisiaealcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels ofATF1andATF2greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. Theatf1Δatf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles ofATF1andATF2led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in theirATFgenes.
Enhanced Production of α-Galactosyl Epitopes by Metabolically Engineered
Pichia pastoris
Authors: Shao Jun , Hayashi Takahisa , Wang Peng George
Published: September 2003

A metabolically engineered
Pichia pastori
s strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from
Vigna radiata
, a truncated UDP-glucose C4 epimerase from
Saccharomyces cerevisiae
, and a truncated bovine α-1,3-galactosyltransferase. Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of
P. pastori
s strain GS115. The proteins were coexpressed intracellularly under the induction of methanol. After permeabilization, the whole
P. pastori
s cells were used to synthesize α-galactosyl (α-Gal) trisaccharide (Galα1,3Galβ1,4Glc) with in situ regeneration of UDP-galactose. Up to 28 mM α-Gal was accumulated in a 200-ml reaction. The
Pichia
system described here is simple and flexible. This work demonstrates that recombinant
P. pastori
s is an excellent alternative to
Escherichia coli
transformants in large-scale synthesis of oligosaccharides.
Prevalence of
Escherichia coli
O157 in Cattle Feeds in Midwestern Feedlots
Authors: Dodd Charles C. , Sanderson Michael W. , Sargeant Jan M. , Nagaraja T. G. , Oberst Richard D. , Smith Robert A. , Griffin D. Dee
Published: September 2003

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with
Escherichia coli
O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting
E. coli
O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of
E. coli
O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of
E. coli
O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the
eaeA
gene, and the presence of at least one Shiga toxin.
E. coli
O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin.
E. coli
O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between
E. coli
O157 prevalence and generic coliform counts in feeds. The prevalence of
E. coli
O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of
E. coli
O157 in order to develop strategies to prevent food-borne disease in humans.
Isolation of Strains Belonging to the Cosmopolitan
Polynucleobacter necessarius
Cluster from Freshwater Habitats Located in Three Climatic Zones
Authors: Hahn Martin W.
Published: September 2003

More than 40 bacterial strains belonging to the cosmopolitan
Polynucleobacter necessarius
cluster (
Betaproteobacteria
) were isolated from a broad spectrum of freshwater habitats located in three climatic zones. Sequences affiliated with the freshwater
P. necessarius
cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems. Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far. The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method. The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods. The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates. Thus, the previous lack of isolates cannot be explained by a lack of appropriate media. Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 μm
3
), even when grown in medium containing high concentrations of organic substances. Thus, these strains are obligate ultramicrobacteria. The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (
Actinobacteria
) and the SAR11 cluster strains (
Alphaproteobacteria
).
Nitrilotriacetate Stimulation of Anaerobic Fe(III) Respiration by Mobilization of Humic Materials in Soil
Authors: Luu Y. , Ramsay B. A. , Ramsay J. A.
Published: September 2003

An enrichment culture capable of naphthalene mineralization reduced Fe(III) oxides without direct contact in anaerobic soil microcosms when the Fe(III) was placed in dialysis membranes or entrapped within alginate beads. Both techniques demonstrated that a component in soil, possibly humic materials, facilitated Fe(III) reduction when direct contact between cells and Fe(III) was not possible. The addition of the synthetic Fe(III) chelator, nitrilotriacetic acid (NTA), to soil enhanced Fe(III) reduction across the dialysis membrane and alginate beads, with the medium changing from clear to a dark brown color. An NTA-soil extract was more effective in Fe(III) reduction than the extracted soil itself. Characteristics of the NTA extract were consistent with that of humic substances. The results indicate that NTA improved Fe(III) reduction not by Fe(III) solubilization but by extraction of humic substances from soil into the aqueous medium. This is the first study in which stimulation of Fe(III) reduction through the addition of chemical chelators is shown to be due to the extraction of electron-shuttling compounds from the soil and not to solubilization of the Fe(III) and indicates that mobilization of humic materials could be an important component of anaerobic biostimulation.
Waterborne Outbreak of Gastroenteritis Associated with a Norovirus
Authors: Parshionikar Sandhya U. , Willian-True Sandra , Fout G. Shay , Robbins David E. , Seys Scott A. , Cassady Joslyn D. , Harris Richard
Published: September 2003

The Wyoming Department of Health investigated an outbreak of acute gastroenteritis among persons who dined at a tourist saloon in central Wyoming during October 2001. Human caliciviruses (HuCVs) were suspected as the etiological agent of the outbreak based on the incubation period, duration of illness, and symptoms observed in ill patrons. A retrospective cohort study demonstrated that ill patrons were 4.5 times more likely to have exposure to drinking water and/or ice than nonill patrons. No food items were associated with illness. An environmental investigation gave evidence that the saloon's groundwater was contaminated with sewage. Water from the saloon's only well was processed for viruses. The processed water sample and stool samples collected from three ill patrons were analyzed by reverse transcription-PCR (RT-PCR) for the presence of HuCV. All positive RT-PCR results were confirmed by sequence and phylogenetic analyses of cloned RT-PCR products. A genogroup I, subtype 3, HuCV stain was found to be present in the well water sample and two stool samples. In addition, a genogroup II, subtype 6, strain was detected in one stool sample. The identification of the same HuCV strain in both the well water and stool samples strongly suggests a link between exposure to well water and the outbreak of gastroenteritis. The presence of a genogroup II, subtype 6, strain in one of the stool samples suggests that multiple HuCV strains may have been involved in this outbreak. The laboratory isolation of HuCV strains from outbreak-associated drinking water is relatively novel in the United States. This investigation outlines the procedure for virus isolation and illustrates the utility of RT-PCR for the identification of HuCV in large volumes of water and stool samples obtained during outbreaks of acute nonbacterial gastroenteritis.
Diversity of
Bacillus thuringiensis
Strains from Latin America with Insecticidal Activity against Different Mosquito Species
Authors: Ibarra Jorge E. , del Rincón M. Cristina , Ordúz Sergio , Noriega David , Benintende Graciela , Monnerat Rose , Regis Leda , de Oliveira Cláudia M. F. , Lanz Humberto , Rodriguez Mario H. , Sánchez Jorge , Peña Guadalupe , Bravo Alejandra
Published: September 2003

The characterization of selected
Bacillus thuringiensis
strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against
Aedes aegypti
,
Culex quinquefasciatus
, and
Anopheles albimanus
larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for
cry
and
cyt
genes encoding proteins active against mosquitoes (
cyt1
,
cyt2
,
cry2
,
cry4A
,
cry4B
,
cry10
,
cry11
,
cry17
,
cry19
,
cry24
,
cry25
,
cry27
,
cry29
,
cry30
,
cry32
,
cry39
, and
cry40
). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against
A
.
aegypti
and
C
.
quinquefasciatus
larvae than
B
.
thuringiensis
subsp.
israelensis
and displayed high similarities with the
B
.
thuringiensis
subsp.
israelensis
used in this study with regard to protein and plasmid profiles and the presence of
cry
genes. Strain 147-8906 has activity against
A
.
aegypti
similar to that of
B
.
thuringiensis
subsp.
israelensis
but has different protein and plasmid profiles. This strain, harboring
cry11
,
cry30
,
cyt1
, and
cyt2
genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel
cry11B
gene.
Involvement of a Sialic Acid-Binding Lectin with Hemagglutination and Hydrophobicity of
Flavobacterium psychrophilum
Authors: Møller Jeannette Dan , Larsen Jens Laurits , Madsen Lone , Dalsgaard Inger
Published: September 2003

Strains of
Flavobacterium psychrophilum
were studied for their ability to adhere and cause agglutination of erythrocytes and yeast cells. Strains of the serotype Th showed low or no hemagglutinating (HA) properties toward human, avian, bovine, and rainbow trout erythrocytes, whereas strains of serotype Fd and Fp
T
exhibited distinct HA properties. None of the strains was able to cause agglutination of yeast cells. Greater adherence specificity toward rainbow trout blood cells was seen for the HA-positive strains. Growth at 5°C, compared to that at 15°C, induced an increase in the hemagglutination of some strains. HA activities of
F. psychrophilum
were inhibited only by sialic acid (
N
-acetyl-neuraminic acid), heat treatment at 65°C, and proteinase K treatment and not by any of seven other carbohydrates, periodate oxidation, or treatment with trypsin. The supernatant from washed bacterial cells also showed some HA properties. All strains were shown to be highly hydrophobic by the hydrophobic interaction chromatography test, although some contradictions to the results of the salt aggregation test (showing some strains as less hydrophobic) were seen. These results indicate that the aggregation of
F. psychrophilum
and erythrocytes depend on a lectin present on the surface of HA-positive
F. psychrophilum
strains and absent on HA-negative strains. This lectin reacts specifically with sialic acid. The adhesion differences observed for
F. psychrophilum
strains do not appear to correlate with the virulence but still provide insights into the interaction of
F. psychrophilum
and rainbow trout.
Does Virus-Induced Lysis Contribute Significantly to Bacterial Mortality in the Oxygenated Sediment Layer of Shallow Oxbow Lakes?
Authors: Fischer Ulrike R. , Wieltschnig Claudia , Kirschner Alexander K. T. , Velimirov Branko
Published: September 2003

Despite the recognition that viruses are ubiquitous components of aquatic ecosystems, the number of studies on viral abundance and the ecological role of viruses in sediments is scarce. In this investigation, the interactions between viruses and bacteria were studied in the oxygenated silty sediment layer of a mesotrophic oxbow lake. A long-term study (13 months) and a diel study revealed that viruses are a numerically important and dynamic component of the microbial community. The abundance and decay rates ranged from 4.3 × 10
9
to 7.2 × 10
9
particles ml of wet sediment
−1
and from undetectable to 22.2 × 10
7
particles ml
−1
h
−1
, respectively, and on average the values were 2 orders of magnitude higher than the values for the overlying water. In contrast to our expectations, viruses did not contribute significantly to the bacterial mortality in the sediment, since on average only 6% (range, 0 to 25%) of the bacterial secondary production was controlled by viruses. The low impact of viruses on the bacterial community may be associated with the quantitatively low viral burden that benthic bacteria have to cope with compared to the viral burden with which bacterial assemblages in the water column are confronted. The virus-to-bacterium ratio of the sediment varied between 0.9 and 3.2, compared to a range of 5.0 to 12.4 obtained for the water column. We speculate that despite high numbers of potential hosts, the possibility of encountering a host cell is limited by the physical conditions in the sediment, which is therefore not a favorable environment for viral proliferation. Our data suggest that viruses do not play an important role in the processing and transfer of bacterial carbon in the oxygenated sediment layer of the environment investigated.
Effect of Acidic pH on Expression of Surface-Associated Proteins of
Streptococcus oralis
Authors: Wilkins Joanna C. , Beighton David , Homer Karen A.
Published: September 2003

Streptococcus oralis
, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of
S. oralis
and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of
Streptococcus pneumoniae
. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from
Lactococcus lactis
, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.
Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from
Lactobacillus helveticus
PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species
Authors: Minervini F. , Algaron F. , Rizzello C. G. , Fox P. F. , Monnet V. , Gobbetti M.
Published: September 2003

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of
Lactobacillus helveticus
PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α
S1
-casein (α
S1
-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α
S1
-CN f1-6 and α
S2
-CN f182-185 and f186-188; caprine β-CN f58-65 and α
S2
-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (
S
)-
N
-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC
50
) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC
50
s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as
Enterococcus faecium
,
Bacillus megaterium
,
Escherichia coli
,
Listeria innocua
,
Salmonella
spp.,
Yersinia enterocolitica
, and
Staphylococcus aureus
. The MIC for
E. coli
F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of
L. helveticus
PR4 or by trypsin and chymotrypsin.
Population Genetics of the Nomenspecies
Enterobacter cloacae
Authors: Hoffmann Harald , Roggenkamp Andreas
Published: September 2003

The genetic heterogeneity of the nomenspecies
Enterobacter cloacae
is well known.
Enterobacter asburiae
,
Enterobacter cancerogenus
,
Enterobacter dissolvens
,
Enterobacter hormaechei
,
Enterobacter kobei
, and
Enterobacter nimipressuralis
are closely related to it and are subsumed in the so-called
E. cloacae
complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies
E. cloacae
, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71
R
were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes
hsp60
,
rpoB
, and
hemB
as well as
ampC
, the gene of a class C β-lactamase. Based on the neighbor-joining tree of the
hsp60
sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of
rpoB
and
hemB
sequences and
ampC
PCR-RFLPs. Sequence crowd xiii split into two groups after
rpoB
analysis. Only three strains formed a cluster with the type strain of
E. cloacae
, indicating that the minority of isolates identified as
E. cloacae
truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the
E. cloacae
complex.
Novel Derivatives of 9,10-Anthraquinone Are Selective Algicides against the Musty-Odor CyanobacteriumOscillatoria perornata
Authors: Schrader Kevin K. , Nanayakkara N. P. Dhammika , Tucker Craig S. , Rimando Agnes M. , Ganzera Markus , Schaneberg Brian T.
Published: September 2003

Musty “off-flavor” in pond-cultured channel catfish (Ictalurus punctatus) costs the catfish production industry in the United States at least $30 million annually. The cyanobacteriumOscillatoria perornata(Skuja) is credited with being the major cause of musty off-flavor in farm-raised catfish in Mississippi. The herbicides diuron and copper sulfate, currently used by catfish producers as algicides to help mitigate musty off-flavor problems, have several drawbacks, including broad-spectrum toxicity towards the entire phytoplankton community that can lead to water quality deterioration and subsequent fish death. By use of microtiter plate bioassays, a novel group of compounds derived from the natural compound 9,10-anthraquinone have been found to be much more selectively toxic towardsO. perornatathan diuron and copper sulfate. In efficacy studies using limnocorrals placed in catfish production ponds, application rates of 0.3 μM (125 μg/liter) of the most promising anthraquinone derivative, 2-[methylamino-N-(1′-methylethyl)]-9,10-anthraquinone monophosphate (anthraquinone-59), dramatically reduced the abundance ofO. perornataand levels of 2-methylisoborneol, the musty compound produced byO. perornata. The abundance of green algae and diatoms increased dramatically 2 days after application of a 0.3 μM concentration of anthraquinone-59 to pond water within the limnocorrals. The half-life of anthraquinone-59 in pond water was determined to be 19 h, making it much less persistent than diuron. Anthraquinone-59 appears to be promising for use as a selective algicide in catfish aquaculture.
Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci
Authors: Wernérus Henrik , Samuelson Patrik , Ståhl Stefan
Published: September 2003

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for
Staphylococcus carnosus
.
Staphylococcus aureus
protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on
S. carnosus
cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.
Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates
Authors: Furushita Manabu , Shiba Tsuneo , Maeda Toshimichi , Yahata Megumi , Kaneoka Azusa , Takahashi Yukinori , Torii Keizo , Hasegawa Tadao , Ohta Michio
Published: September 2003

Tetracycline-resistant (Tet
r
) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet
r
gram-negative strains, 29 were identified as carrying
tetB
only. Four carried
tetY
, and another four carried
tetD
. Three strains carried
tetC
, two strains carried
tetB
and
tetY
, and one strain carried
tetC
and
tetG
. Sequence analyses indicated the identity in Tet
r
genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for
tetB
, 98.2 to 100% for
tetC
, 99.7 to 100% for
tetD
, 92.0 to 96.2% for
tetG
, and 97.1 to 100% for
tetY
. Eleven of the Tet
r
strains transferred Tet
r
genes by conjugation to
Escherichia coli
HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of
Photobacterium
,
Vibrio
,
Pseudomonas
,
Alteromonas
,
Citrobacter
, and
Salmonella
spp., and they transferred
tetB
,
tetY
, or
tetD
to the recipients. Because NaCl enhanced their growth, these Tet
r
strains, except for the
Pseudomonas
,
Citrobacter
, and
Salmonella
strains, were recognized as marine bacteria. Our results suggest that
tet
genes from fish farm bacteria have the same origins as those from clinical strains.
Introduction of
Culex
Toxicity into
Bacillus thuringiensis
Cry4Ba by Protein Engineering
Authors: Abdullah Mohd Amir F. , Alzate Oscar , Mohammad Marwan , McNall Rebecca J. , Adang Michael J. , Dean Donald H.
Published: September 2003

Bacillus thuringiensis
mosquitocidal toxin Cry4Ba has no significant natural activity against
Culex quinquefasciatus
or
Culex pipiens
(50% lethal concentrations [LC
50
], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes,
Aedes aegypti
,
Anopheles quadrimaculatus
,
C
.
quinquefasciatus
, and
C
.
pipiens
. Putative loop 1 and 2 exchanges eliminated activity towards
A
.
aegypti
and
A
.
quadrimaculatus
. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against
C
.
quinquefasciatus
(LC
50
≅ 114 ng/ml) and
C
.
pipiens
(LC
50
⩬ 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against
Anopheles
or
Aedes
. These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing
B
.
thuringiensis
Cry toxins.
Characterization of Microbial Communities in Gas Industry Pipelines
Authors: Zhu Xiang Y. , Lubeck John , Kilbane John J.
Published: September 2003

Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines. Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed. A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of
Proteobacteria
and gram-positive bacteria. The most frequently encountered bacterial species was
Comamonas denitrificans
, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion. The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I,
Methanobacteriales
; order III,
Methanomicrobiales
; and order IV,
Methanosarcinales
. Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion.
Morphological, Host Range, and Genetic Characterization of Two Coliphages
Authors: Goodridge Lawrence , Gallaccio Alicia , Griffiths Mansel W.
Published: September 2003

Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to theMyoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains ofEscherichia coliand other enterobacteria. The results showed that both phages could infect many serotypes ofE.coli. Among the enterobacteria,Proteus mirabilis,Shigella dysenteriae, and twoSalmonellastrains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence ofE.coliin animals and food.
Effect of
Campylobacter
-Specific Maternal Antibodies on
Campylobacter jejuni
Colonization in Young Chickens
Authors: Sahin Orhan , Luo Naidan , Huang Shouxiong , Zhang Qijing
Published: September 2003

Using laboratory challenge experiments, we examined whether
Campylobacter-
specific maternal antibody (MAB) plays a protective role in young chickens, which are usually free of
Campylobacter
under natural production conditions. Kinetics of
C. jejuni
colonization were compared by infecting 3-day-old broiler chicks, which were naturally positive for
Campylobacter
-specific MAB, and 21-day-old broilers, which were negative for
Campylobacter
-specific MAB. The onset of colonization occurred much sooner in birds challenged at the age of 21 days than it did in the birds inoculated at 3 days of age, which suggested a possible involvement of specific MAB in the delay of colonization. To further examine this possibility, specific-pathogen-free layer chickens were raised under laboratory conditions with or without
Campylobacter
infection, and their 3-day-old progenies with (MAB
+
) or without (MAB
−
)
Campylobacter
-specific MAB were orally challenged with
C. jejuni
. Significant decreases in the percentage of colonized chickens were observed in the MAB
+
group during the first week compared with the MAB
−
group. These results indicate that
Campylobacter-
specific MAB plays a partial role in protecting young chickens against colonization by
C. jejuni
. Presence of MAB in young chickens did not seem to affect the development of systemic immune response following infection with
C. jejuni
. However, active immune responses to
Campylobacter
occurred earlier and more strongly in birds infected at 21 days of age than those infected at 3 days of age. Clearance of
Campylobacter
infection was also observed in chickens infected at 21 days of age. Taken together, these findings (i) indicate that anti-
Campylobacter
MAB contributes to the lack of
Campylobacter
infection in young broiler chickens in natural environments and (ii) provide further evidence supporting the feasibility of development of immunization-based approaches for control of
Campylobacter
infection in poultry.
Assessment of Methods for Detection of Infectious
Cryptosporidium
Oocysts and
Giardia
Cysts in Reclaimed Effluents
Authors: Quintero-Betancourt W. , Gennaccaro A. L. , Scott T. M. , Rose J. B.
Published: September 2003

This study evaluates the occurrence of
Cryptosporidium
oocysts and
Giardia
cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.
18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates
Authors: Wu Zhihong , Tsumura Yoshihiko , Blomquist Göran , Wang Xiao-Ru
Published: September 2003

In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between
Zygomycota
and
Ascomycota
. Within
Ascomycota
, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi.
Wallemia
appeared as the group most diverged from all the other
Ascomycota
species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.
Hydroxycinnamate (
hca
) Catabolic Genes from
Acinetobacter
sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters
Authors: Parke Donna , Ornston L. Nicholas
Published: September 2003

Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium
Acinetobacter
sp. strain ADP1. Genes for protocatechuate catabolism are central to the
dca-pca-qui-pob-hca
chromosomal island, for which gene designations corresponding to catabolic function are
dca
(dicarboxylic acid),
pca
(protocatechuate),
qui
(quinate),
pob
(
p
-hydroxybenzoate), and
hca
(hydroxycinnamate).
Acinetobacter hcaC
had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate,
p
-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from
pobS
to an
Xba
I site 5.3 kb beyond
hcaC
was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the
hca
region of the chromosome. pZR8200 enabled
Escherichia coli
to convert
p
-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated
hcaA
,
hcaB
,
hcaK
,
hcaR
, and ORF1. An
Acinetobacter
strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with
p
-coumarate. A chromosomal fusion of
lacZ
to the
hcaE
gene was used to monitor expression of the
hcaABCDE
promoter. LacZ was induced over 100-fold by growth in the presence of caffeate,
p
-coumarate, or ferulate. The protein deduced to be encoded by
hcaR
shares 28% identity with the aligned
E. coli
repressor, MarR. A knockout of
hcaR
produced a constitutive phenotype, as assessed in the
hcaE
::
lacZ
-Km
r
genetic background, revealing HcaR to be a repressor as well. Expression of
hcaE
::
lac
Z in strains with knockouts in
hcaA
,
hcaB
, or
hcaC
revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the
hcaABCDE
genes by HcaR.
The Metabolic Pathway of 4-Aminophenol in
Burkholderia
sp. Strain AK-5 Differs from That of Aniline and Aniline with C-4 Substituents
Authors: Takenaka Shinji , Okugawa Susumu , Kadowaki Maho , Murakami Shuichiro , Aoki Kenji
Published: September 2003

Burkholderia
sp. strain AK-5 utilized 4-aminophenol as the sole carbon, nitrogen, and energy source. A pathway for the metabolism of 4-aminophenol in strain AK-5 was proposed based on the identification of three key metabolites by gas chromatography-mass spectrometry analysis. Strain AK-5 converted 4-aminophenol to 1,2,4-trihydroxybenzene via 1,4-benzenediol. 1,2,4-Trihydroxybenzene 1,2-dioxygenase cleaved the benzene ring of 1,2,4-trihydroxybenzene to form maleylacetic acid. The enzyme showed a high dioxygenase activity only for 1,2,4-trihydroxybenzene, with

K
m

and
V
max
values of 9.6 μM and 6.8 μmol min
−1
mg of protein
−1
, respectively.
Mercury Methylation Independent of the Acetyl-Coenzyme A Pathway in Sulfate-Reducing Bacteria
Authors: Ekstrom Eileen B. , Morel François M. M. , Benoit Janina M.
Published: September 2003

Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain,
Desulfovibrio desulfuricans
LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two
Desulfobacter
strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B
12
in some and perhaps many incomplete-oxidizing SRB strains.
Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System ofStreptococcus thermophilus
Authors: Cochu Armelle , Vadeboncoeur Christian , Moineau Sylvain , Frenette Michel
Published: September 2003

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIABMan, two proteins involved in regulatory mechanisms. While most strains ofStreptococcus thermophilusdo not or poorly metabolize glucose, compelling evidence suggests thatS. thermophiluspossesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. Theptsoperon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kbptsHmRNA and a 2.3-kbptsHImRNA. The specific glucose/mannose PTS proteins, IIABMan, IICMan, IIDMan, and the ManO protein, are encoded bymanL,manM,manN, andmanO, respectively, which make up themanoperon. Themanoperon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIABManas well as membrane fragments containing IICManand IIDMan. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of theS. thermophilusPTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that inS. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIABMan.