bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Genetic Characterization of
dca
Genes Required for β-Oxidation of Straight-Chain Dicarboxylic Acids in
Acinetobacter
sp. Strain ADP1
Authors: Parke D. , Garcia M. A. , Ornston L. N. 
Published: July 2002

ABSTRACT

A previous study of deletions in the protocatechuate (
pca
) region of the
Acinetobacter
sp. strain ADP1 chromosome revealed that genes required for utilization of the six-carbon dicarboxylic acid, adipic acid, are linked to the
pca
structural genes. To investigate the genes involved in adipate catabolism, a 33.8-kb
Sac
I fragment, which corrects a deletion spanning this region, was cloned. In addition to containing known
pca
,
qui
, and
pob
genes (for protocatechuate, quinate, and 4-hydroxybenzoate dissimilation), clone pZR8000 contained 10 kb of DNA which was the subject of this investigation. A mutant strain of
Escherichia coli
DH5α, strain EDP1, was isolated that was able to utilize protocatechuate and 4-hydroxybenzoate as growth substrates when EDP1 cells contained pZR8000. Sequence analysis of the new region of DNA on pZR8000 revealed open reading frames predicted to be involved in β-oxidation. Knockouts of three genes implicated in β-oxidation steps were introduced into the chromosome of
Acinetobacter
sp. strain ADP1. Each of the mutants was unable to grow with adipate. Because the mutants were affected in their ability to utilize additional saturated, straight-chain dicarboxylic acids, the newly discovered 10 kb of DNA was termed the
dca
(dicarboxylic acid) region. Mutant strains included one with a deletion in
dcaA
(encoding an acyl coenzyme A [acyl-CoA] dehydrogenase homolog), one with a deletion in
dcaE
(encoding an enoyl-CoA hydratase homolog), and one with a deletion in
dcaH
(encoding a hydroxyacyl-CoA dehydrogenase homolog). Data on the
dca
region should help us probe the functional significance and interrelationships of clustered genetic elements in this section of the
Acinetobacter
chromosome.
Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from
Pyrococcus horikoshii
OT3
Authors: Ishikawa Kazuhiko , Ishida Hiroyasu , Matsui Ikuo , Kawarabayasi Yutaka , Kikuchi Hisasi 
Published: July 2002

ABSTRACT

Genome sequencing of the thermophilic archaeon
Pyrococcus horikoshii
OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase of
Sulfolobus solfataricus
and also to that encoding the aminoacylase from
Bacillus stearothermophilus
. The gene from
P. horikoshii
comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in
Escherichia coli
with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.
Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning
Authors: Qiu Xiaoyun , Wu Liyou , Huang Heshu , McDonel Patrick E. , Palumbo Anthony V. , Tiedje James M. , Zhou Jizhong 
Published: July 2002

ABSTRACT

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division
Proteobacteria
as well as gram-positive bacterium, all of which could be distinguished by
Hha
I restriction digestion patterns. The overall PCR artifacts were significantly different among the three
Taq
DNA polymerases examined: 20% for Z-
Taq
, with the highest processitivity; 15% for LA-
Taq
, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, Ampli
Taq
. In contrast to the theoretical prediction, the frequency of chimeras for both Z-
Taq
(8.7%) and LA-
Taq
(6.2%) was higher than that for Ampli
Taq
(2.5%). The frequencies of chimeras and of heteroduplexes for Z-
Taq
were almost three times higher than those of Ampli
Taq
. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using Ampli
Taq
and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.
Enhanced Iron Uptake ofSaccharomyces cerevisiaeby Heterologous Expression of a Tadpole Ferritin Gene
Authors: Shin Young-Mi , Kwon Tae-Ho , Kim Kyung-Suk , Chae Keon-Sang , Kim Dae-Hyuk , Kim Jae-Ho , Yang Moon-Sik 
Published: July 2002

ABSTRACTWe genetically engineeredSaccharomyces cerevisiaeto express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene(TFH)into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody againstEscherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 μg per gram (dry cell weight) of the recombinant yeast but was 210 μg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.
Genetic Evidence that Loss of Virulence Associated with
gacS
or
gacA
Mutations in
Pseudomonas syringae
B728a Does Not Result from Effects on Alginate Production
Authors: Willis David K. , Holmstadt Jeremy J. , Kinscherf Thomas G. 
Published: July 2002

ABSTRACT

Mutations in the global regulatory genes
gacS
and
gacA
render
Pseudomonas syringae
pv. syringae strain B728a completely nonpathogenic in foliar infiltration assays on bean plants. It had been previously demonstrated that
gac
genes regulate alginate production in
Pseudomonas
species, while other published work indicated that alginate is involved in the pathogenic interaction of
P. syringae
on bean plants. Together, these results suggested that the effects of
gacS
and
gacA
mutations on virulence in B728a might stem directly from a role in regulating alginate. In this report, we confirm a role for
gac
genes in both
algD
expression and alginate production in B728a. However, B728a mutants completely devoid of detectable alginate were as virulent as the wild-type strain in our assay. Thus, factors other than, or in addition to, a deficiency of alginate must be involved in the lack of pathogenicity observed with
gacS
and
gacA
mutants.
Characterization of Glycine Sarcosine
N
-Methyltransferase and Sarcosine Dimethylglycine
N
-Methyltransferase
Authors: Nyyssölä Antti , Reinikainen Tapani , Leisola Matti 
Published: July 2002

ABSTRACT

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine
N
-methyltransferase (GSMT) and sarcosine dimethylglycine
N
-methyltransferase (SDMT) of
Ectothiorhodospira halochloris
catalyze the threefold methylation of glycine to betaine, with
S
-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in
Escherichia coli
and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent

K
m

and
V
max
values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product
S
-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.
Predator-Specific Enrichment of Actinobacteria from a Cosmopolitan Freshwater Clade in Mixed Continuous Culture
Authors: Pernthaler Jakob , Posch Thomas , S̆imek Karel , Vrba Jaroslav , Pernthaler Annelie , Glöckner Frank Oliver , Nübel Ulrich , Psenner Roland , Amann Rudolf 
Published: July 2002

ABSTRACT

We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyte
Cryptomonas
sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (
Ochromonas
sp.) always resulted in an immediate bloom of a single phylotype population of members of the class
Actinobacteria
(Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate
Cyclidium glaucoma
. However, when the
Ochromonas
sp. was added after the establishment of
C. glaucoma
, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an
Aquabacterium
sp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.
Inactivation of
Mycobacterium paratuberculosis
by Pulsed Electric Fields
Authors: Rowan Neil J. , MacGregor Scott J. , Anderson John G. , Cameron Douglas , Farish Owen 
Published: July 2002

ABSTRACT

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of
Mycobacterium paratuberculosis
cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50°C (2,500 pulses at 30 kV/cm) reduced the level of viable
M. paratuberculosis
cells by approximately 5.3 and 5.9 log
10
CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of
M. paratuberculosis
at lower temperatures resulted in less lethality. Heating alone at 50°C for 25 min or at 72°C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of
M. paratuberculosis
of approximately 0.01 and 2.4 log
10
CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to
M. paratuberculosis
.
Detoxification of Corn Antimicrobial Compounds as the Basis for Isolating
Fusarium verticillioides
and Some Other
Fusarium
Species from Corn
Authors: Glenn A. E. , Hinton D. M. , Yates I. E. , Bacon C. W. 
Published: July 2002

ABSTRACT

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively.
Fusarium verticillioides
(=
F. moniliforme
) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29
Fusarium
species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were
F. verticillioides
,
F. subglutinans
,
F. cerealis
(=
F. crookwellense
), and
F. graminearum
. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of
F. verticillioides
from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of
F. verticillioides
infection in field samples.
Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic
Escherichia coli
Groups
Authors: Grant Michael A. , Weagant Stephen D. , Feng Peter 
Published: July 2002

ABSTRACT

The enzyme glutamate decarboxylase (GAD) is prevalent in
Escherichia coli
but few strains in the various pathogenic
E. coli
groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the
gadA
and
gadB
genes encoding GAD, we examined the distribution of the
gadAB
genes among enteric bacteria. Analysis of 173 pathogenic
E. coli
strains, including 125 enterohemorrhagic
E. coli
isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic
E. coli
, enterotoxigenic
E. coli
, enteroinvasive
E. coli
, and other Shiga toxin-producing
E. coli
(STEC) serotypes, showed that
gadAB
genes were present in all these strains. Among the 22 non-
E. coli
isolates tested, only the 6
Shigella
spp. carried
gadAB
. Analysis of naturally contaminated water and food samples using a
gadAB
-specific DNA probe that was labeled with digoxigenin showed that a
gadAB
-based assay is as reliable as standard methods that enumerate
E. coli
organisms on the basis of lactose fermentation. The presence of few
E. coli
cells initially seeded into produce rinsates could be detected by PCR to
gadA/B
genes after overnight enrichment. A multiplex PCR assay using the
gadAB
primers in combination with primers to Shiga toxin (Stx) genes
stx
1
and
stx
2
was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The
gadAB
primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic
E. coli
groups.
True Chemotaxis in Oxygen Gradients of the Sulfur-Oxidizing Bacterium
Thiovulum majus
Authors: Thar Roland , Fenchel Tom 
Published: July 2002

ABSTRACT

Observations of free-swimming
Thiovulum majus
cells show that these bacteria exhibit a phobic response as well as true chemotaxis in oxygen gradients. Both phenomena of their chemotactic behavior are integrated into a single model of helical klinotaxis, which is demonstrated by computer simulations.
l
-Pyroglutamate Spontaneously Formed from
l
-Glutamate Inhibits Growth of the Hyperthermophilic Archaeon
Sulfolobus solfataricus
Authors: Park Chan B. , Lee Sun Bok , Ryu Dewey D. Y. 
Published: July 2002

ABSTRACT

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of
Sulfolobus solfataricus
in medium containing
l
-glutamate, we observed formation of
l
-pyroglutamic acid (PGA). PGA formed spontaneously from
l
-glutamate under culture conditions (78°C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of
S. solfataricus
growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of
l
-glutamate or
l
-aspartate to the medium. PGA was also produced from the
l
-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78°C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of
l
-glutamate, such as
l
-methionine sulfoxide, glutaric acid, succinic acid, and
l
-glutamic acid γ-methyl ester, also inhibited the growth of
S. solfataricus
, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues,
N
-acetyl-
l
-glutamate enhanced the growth of
S. solfataricus.
This compound was stable under cell culture conditions, and replacement of
l
-glutamate with
N
-acetyl-
l
-glutamate in the medium resulted in increased cell density.
Recovery of
Streptococcus iniae
from Diseased Fish Previously Vaccinated with a
Streptococcus
Vaccine
Authors: Bachrach Gilad , Zlotkin Amir , Hurvitz Avshalom , Evans Donald L. , Eldar Avi 
Published: July 2002

ABSTRACT

Streptococcus iniae
was recovered from diseased rainbow trout (
Oncorhynchus mykiss
, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.
Survival of Fecal Coliforms in Dry-Composting Toilets
Authors: Redlinger Thomas , Graham Jay , Corella-Barud Verónica , Avitia Raquel 
Published: July 2002

ABSTRACT

The dry-composting toilet, which uses neither water nor sewage infrastructure, is a practical solution in areas with inadequate sewage disposal and where water is limited. These systems are becoming increasingly popular and are promoted to sanitize human excreta and to recycle them into fertilizer for nonedible plants, yet there are few data on the safety of this technology. This study analyzed fecal coliform reduction in approximately 90 prefabricated, dry-composting toilets (Sistema Integral de Reciclamiento de Desechos Orgánicos [SIRDOs]) that were installed on the U.S.-Mexico border in Ciudad Juárez, Chihuahua, Mexico. The purpose of this study was to determine fecal coliform reduction over time and the most probable method of this reduction. Biosolid waste samples were collected and analyzed at approximately 3 and 6 months and were classified based on U.S. Environmental Protection Agency standards. Results showed that class A compost (high grade) was present in only 35.8% of SIRDOs after 6 months. The primary mechanism for fecal coliform reduction was found to be desiccation rather than biodegradation. There was a significant correlation (
P
= 0.008) between classification rating and percent moisture categories of the biosolid samples: drier samples had a greater proportion of class A samples. Solar exposure was critical for maximal class A biosolid end products (
P
= 0.001). This study only addressed fecal coliforms as an indicator organism, and further research is necessary to determine the safety of composting toilets with respect to other pathogenic microorganisms, some of which are more resistant to desiccation.
Molecular Characterization of Novel Red Green Nonsulfur Bacteria from Five Distinct Hot Spring Communities in Yellowstone National Park
Authors: Boomer Sarah M. , Lodge Daniel P. , Dutton Bryan E. , Pierson Beverly 
Published: July 2002

ABSTRACT

We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60°C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll
a
(Bchl
a
), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl
a
protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to
Roseiflexus castenholzii
, a red filamentous bacterium from Japan that also contains only Bchl
a
. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new “red” cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to
Roseiflexus
and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl
a
spectrum differences or obvious features of the habitat.