bluuebunny/crossref_metadata_2025_split huggingface dataset:
Population Dynamics of
Vibrio fischeri
during Infection of
Euprymna scolopes
Authors: McCann Jessica , Stabb Eric V. , Millikan Deborah S. , Ruby Edward G. 
Published: October 2003

ABSTRACT

The luminous bacterium
Vibrio fischeri
colonizes a specialized light-emitting organ within its squid host,
Euprymna scolopes
. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of
V. fischeri
during colonization more fully, we used mini-Tn
7
transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on
V. fischeri
cells carrying the transposon, which inserts in single copy in a specific intergenic region of the
V. fischeri
genome. Strains marked with mini-Tn
7
also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of
V. fischeri
cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of
V. fischeri
were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of
V. fischeri
, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.
Tri16Is Required for Esterification of Position C-8 during Trichothecene Mycotoxin Production byFusarium sporotrichioides
Authors: Peplow Andrew W. , Meek Isaac B. , Wiles Melinda C. , Phillips Timothy D. , Beremand Marian N. 
Published: October 2003

ABSTRACTWe previously characterizedTri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis inFusarium sporotrichioidesNRRL 3299. Sequence analysis of the region surroundingTri1revealed a gene, namedTri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption ofTri16were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate thatTri16encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of aTri16ortholog inGibberella pulicarisR-6380 that is likely linked to a presumably inactive ortholog forTri1.
Utilization of Acyl-Homoserine Lactone Quorum Signals for Growth by a Soil Pseudomonad and
Pseudomonas aeruginosa
PAO1
Authors: Huang Jean J. , Han Jong-In , Zhang Lian-Hui , Leadbetter Jared R. 
Published: October 2003

ABSTRACT

Acyl-homoserine lactones (AHLs) are employed by several
Proteobacteria
as quorum-sensing signals. Past studies have established that these compounds are subject to biochemical decay and can be used as growth nutrients. Here we describe the isolation of a soil bacterium,
Pseudomonas
strain PAI-A, that degrades 3-oxododecanoyl-homoserine lactone (3OC12HSL) and other long-acyl, but not short-acyl, AHLs as sole energy sources for growth. The small-subunit rRNA gene from strain PAI-A was 98.4% identical to that of
Pseudomonas aeruginosa
, but the soil isolate did not produce obvious pigments or AHLs or grow under denitrifying conditions or at 42°C. The quorum-sensing bacterium
P. aeruginosa
, which produces both 3OC12HSL and C4HSL, was examined for the ability to utilize AHLs for growth. It did so with a specificity similar to that of strain PAI-A, i.e., degrading long-acyl but not short-acyl AHLs. In contrast to the growth observed with strain PAI-A,
P. aeruginosa
strain PAO1 growth on AHLs commenced only after extremely long lag phases. Liquid-chromatography-atmospheric pressure chemical ionization-mass spectrometry analyses indicate that strain PAO1 degrades long-acyl AHLs via an AHL acylase and a homoserine-generating HSL lactonase. A
P. aeruginosa
gene,
pvdQ
(PA2385), has previously been identified as being a homologue of the AHL acylase described as occurring in a
Ralstonia
species.
Escherichia coli
expressing
pvdQ
catalyzed the rapid inactivation of long-acyl AHLs and the release of HSL.
P. aeruginosa
engineered to constitutively express
pvdQ
did not accumulate its 3OC12HSL quorum signal when grown in rich media. However,
pvdQ
knockout mutants of
P. aeruginosa
were still able to grow by utilizing 3OC12HSL. To our knowledge, this is the first report of the degradation of AHLs by pseudomonads or other γ-
Proteobacteria
, of AHL acylase activity in a quorum-sensing bacterium, of HSL lactonase activity in any bacterium, and of AHL degradation with specificity only towards AHLs with long side chains.
Comparing Denitrification Estimates for a Texas Estuary by Using Acetylene Inhibition and Membrane Inlet Mass Spectrometry
Authors: Bernot Melody J. , Dodds Walter K. , Gardner Wayne S. , McCarthy Mark J. , Sobolev Dmitri , Tank Jennifer L. 
Published: October 2003

ABSTRACTCharacterizing denitrification rates in aquatic ecosystems is essential to understanding how systems may respond to increased nutrient loading. Thus, it is important to ensure the precision and accuracy of the methods employed for measuring denitrification rates. The acetylene (C2H2) inhibition method is a simple technique for estimating denitrification. However, potential problems, such as inhibition of nitrification and incomplete inhibition of nitrous oxide reduction, may influence rate estimates. Recently, membrane inlet mass spectrometry (MIMS) has been used to measure denitrification in aquatic systems. Comparable results were obtained with MIMS and C2H2inhibition methods when chloramphenicol was added to C2H2inhibition assay mixtures to inhibit new synthesis of denitrifying enzymes. Dissolved-oxygen profiles indicated that surface layers of sediment cores subjected to the MIMS flowthrough incubation remained oxic whereas cores incubated using the C2H2inhibition methods did not. Analysis of the microbial assemblages before and after incubations indicated significant changes in the sediment surface populations during the long flowthrough incubation for MIMS analysis but not during the shorter incubation used for the C2H2inhibition method. However, bacterial community changes were also small in MIMS cores at the oxygen transition zone where denitrification occurs. The C2H2inhibition method with chloramphenicol addition, conducted over short incubation intervals, provides a cost-effective method for estimating denitrification, and rate estimates are comparable to those obtained by the MIMS method.
Bacterial Chemotaxis to Naphthalene Desorbing from a Nonaqueous Liquid
Authors: Law Aaron M. J. , Aitken Michael D. 
Published: October 2003

ABSTRACT

Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined. We studied the effect of chemotaxis by
Pseudomonas putida
G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL. Chemotaxis by wild-type
P. putida
G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains. While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source. The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation. The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants.
Community Composition and Functioning of Denitrifying Bacteria from Adjacent Meadow and Forest Soils
Authors: Rich J. J. , Heichen R. S. , Bottomley P. J. , Cromack K. , Myrold D. D. 
Published: October 2003

ABSTRACT

We investigated communities of denitrifying bacteria from adjacent meadow and forest soils. Our objectives were to explore spatial gradients in denitrifier communities from meadow to forest, examine whether community composition was related to ecological properties (such as vegetation type and process rates), and determine phylogenetic relationships among denitrifiers.
nosZ
, a key gene in the denitrification pathway for nitrous oxide reductase, served as a marker for denitrifying bacteria. Denitrifying enzyme activity (DEA) was measured as a proxy for function. Other variables, such as nitrification potential and soil C/N ratio, were also measured. Soil samples were taken along transects that spanned meadow-forest boundaries at two sites in the H. J. Andrews Experimental Forest in the Western Cascade Mountains of Oregon. Results indicated strong functional and structural community differences between the meadow and forest soils. Levels of DEA were an order of magnitude higher in the meadow soils. Denitrifying community composition was related to process rates and vegetation type as determined on the basis of multivariate analyses of
nosZ
terminal restriction fragment length polymorphism profiles. Denitrifier communities formed distinct groups according to vegetation type and site. Screening 225
nosZ
clones yielded 47 unique denitrifying genotypes; the most dominant genotype occurred 31 times, and half the genotypes occurred once. Several dominant and less-dominant denitrifying genotypes were more characteristic of either meadow or forest soils. The majority of
nosZ
fragments sequenced from meadow or forest soils were most similar to
nosZ
from the
Rhizobiaceae
group in α-
Proteobacteria
species. Denitrifying community composition, as well as environmental factors, may contribute to the variability of denitrification rates in these systems.
Identification and Characterization of the
CYP52
Family of
Candida tropicalis
ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids
Authors: Craft David L. , Madduri Krishna M. , Eshoo Mark , Wilson C. Ron 
Published: October 2003

ABSTRACT

Candida tropicalis
ATCC 20336 excretes α,ω-dicarboxylic acids as a by-product when cultured on
n
-alkanes or fatty acids as the carbon source. Previously, a β-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the ω-hydroxylase enzymes catalyzing the first step in the ω-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of ω-oxidation of
n
-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (
CYP52
) of
C. tropicalis
ATCC20336 as well as the accompanying
NCP
were cloned and sequenced. The 10
CYP
genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes,
CYP52A13
and
CYP52A14
showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair
CYP52A17
and
CYP52A18
was also induced under all three conditions but to a lesser extent. Moderate induction of
CYP52A12
was observed. These results identify the
CYP52
and
NCP
genes as being involved in α,ω-dicarboxylic acid production by
C. tropicalis
and provide the foundation for biocatalyst improvement.
Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of
Candida tropicalis
Authors: Eschenfeldt William H. , Zhang Yeyan , Samaha Hend , Stols Lucy , Eirich L. Dudley , Wilson C. Ronald , Donnelly Mark I. 
Published: October 2003

ABSTRACT

Candida tropicalis
ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase.
C. tropicalis
ATCC 20336 contains a family of
CYP
genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C
18:1
), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the
C. tropicalis
cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C
14:0
) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.
Distribution of the Coenzyme M Pathway of Epoxide Metabolism among Ethene- and Vinyl Chloride-Degrading
Mycobacterium
Strains
Authors: Coleman Nicholas V. , Spain Jim C. 
Published: October 2003

ABSTRACT

An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of
Mycobacterium
strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from
Mycobacterium
strain JS60 and the propene oxidizers
Xanthobacter
strain Py2 and
Rhodococcus
strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The
Mycobacterium
EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of
Rhodococcus
strain B-276 than that of
Xanthobacter
strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the
Mycobacterium
strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer.
Lysine-2,3-Aminomutase and β-Lysine Acetyltransferase Genes of Methanogenic Archaea Are Salt Induced and Are Essential for the Biosynthesis of
N
ε
-Acetyl-β-Lysine and Growth at High Salinity
Authors: Pflüger K. , Baumann S. , Gottschalk G. , Lin W. , Santos H. , Müller V. 
Published: October 2003

ABSTRACT

The compatible solute
N
ε
-acetyl-β-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of
N
ε
-acetyl-β-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (
ablA
) and β-lysine acetyltransferase (
ablB
), which are assumed to catalyze
N
ε
-acetyl-β-lysine formation from α-lysine, were identified on the chromosomes of the methanogenic archaea
Methanosarcina mazei
Gö1,
Methanosarcina acetivorans
,
Methanosarcina barkeri
,
Methanococcus jannaschii
, and
Methanococcus maripaludis
. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the
abl
operon) in
M. mazei
Gö1. Expression of the
abl
operon was strictly salt dependent. The
abl
operon was deleted in the genetically tractable
M. maripaludis
. Δ
abl
mutants of
M. maripaludis
no longer produced
N
ε
-acetyl-β-lysine and were incapable of growth at high salt concentrations, indicating that the
abl
operon is essential for
N
ε
-acetyl-β-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.
Optimized Expression of a Thermostable Xylanase from
Thermomyces lanuginosus
in
Pichia pastoris
Authors: Damaso Mônica C. Triches , Almeida Marcius S. , Kurtenbach Eleonora , Martins Orlando B. , Pereira Nei , Andrade Carolina M. M. C. , Albano Rodolpho M. 
Published: October 2003

ABSTRACT

Highly efficient production of a
Thermomyces lanuginosus
IOC-4145 β-1,4-xylanase was achieved in
Pichia pastoris
under the control of the AOX1 promoter.
P. pastoris
colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 2
3
factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in
P. pastoris
makes this expression system attractive for fermentor growth and industrial applications.
Diversity ofWolbachiaEndosymbionts in Heteropteran Bugs
Authors: Kikuchi Yoshitomo , Fukatsu Takema 
Published: October 2003

ABSTRACTAn extensive survey ofWolbachiaendosymbionts in Japanese terrestrial heteropteran bugs was performed by PCR detection with universal primers forwspandftsZgenes ofWolbachia, cloning of the PCR products, restriction fragment length polymorphism analysis of infectingWolbachiatypes, and molecular phylogenetic characterization of all the detectedWolbachiastrains. Of 134 heteropteran species from 19 families examined,Wolbachiainfection was detected in 47 species from 13 families. From the 47 species, 59Wolbachiastrains were identified. Of the 59 strains, 16 and 43 were assigned to A group and B group in theWolbachiaphylogeny, respectively. The 47 species ofWolbachia-infected bugs were classified into 8 species with A infection, 28 species with B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1 species with ABB infection. Molecular phylogenetic analysis showed little congruence betweenWolbachiaphylogeny and host systematics, suggesting frequent horizontal transfers ofWolbachiain the evolutionary course of the Heteroptera. The phylogenetic analysis also revealed several novel lineages ofWolbachia. Based on statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs, interactions between coinfectingWolbachiastrains are generally not intense and thatWolbachiacoinfections have been established through a stochastic process probably depending on occasional horizontal transfers.
Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by
Gluconacetobacter xylinus
I-2281
Authors: Kornmann Henri , Duboc Philippe , Marison Ian , von Stockar Urs 
Published: October 2003

ABSTRACT

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain
Gluconacetobacter xylinus
I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by
G. xylinus.
In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing
G. xylinus
on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.