bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activity of Novispirin G10 againstPseudomonas aeruginosaIn Vitro and in Infected Burns
Authors: Steinstraesser Lars , Tack Brian F. , Waring Alan J. , Hong Teresa , Boo Lee M. , Fan Ming-Hui , Remick Daniel I. , Su Grace L. , Lehrer Robert I. , Wang Stewart C. 
Published: July 2002

ABSTRACTThe emergence of multidrug-resistant microbes has serious implications for managing infection and sepsis and has stimulated efforts to develop alternative treatments, such as antimicrobial peptides. The objective of this study was to test a designer peptide, novispirin G10, against multidrug-resistant microorganisms. By two-stage radial diffusion assays, its activity against such organisms compared favorably with that of standard antibiotics and other antimicrobial peptides. It killed bacteria very rapidly, was nonhemolytic, and was relatively noncytotoxic. The peptide induced an immediate, massive efflux of potassium fromPseudomonas aeruginosa, suggesting that it altered the permeability of its inner membrane. The presence of human serum reduced but did not eliminate its activity. We tested the in vivo activity of novispirin G10 in rats with an infected, partial-thickness burn that covered 20% of their total body surface area. The burned area was seeded with 106CFU of a Silvadene-resistantP. aeruginosastrain, and 24 h later a single treatment with 0, 1, 3, or 6 mg of synthetic novispirin G10 (n= 16 at each concentration) per kg was given intradermally. Significant bacterial killing (P< 0.0001) was evident within 4 h in each peptide group compared to controls receiving vehicle. Antimicrobial peptides such as novispirin G10 may provide a useful alternative or adjunct to standard antibiotic agents in treating burns or other wound infections.
Adaptation of the Nisin-Controlled Expression System in
Lactobacillus plantarum
: a Tool To Study In Vivo Biological Effects
Authors: Pavan Sonia , Hols Pascal , Delcour Jean , Geoffroy Marie-Claude , Grangette Corinne , Kleerebezem Michiel , Mercenier Annick 
Published: July 2002

ABSTRACT

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in
Lactobacillus plantarum
NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by
Lactococcus lactis
. The original two-plasmid NICE system turned out to be poorly suited to
L. plantarum
. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (β-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal
nisRK
regulatory genes were integrated into the chromosome of
L. plantarum
NCIMB8826. Moreover, recombinant
L. plantarum
producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.
Pacific Northwest Marine Sediments Contain Ammonia-Oxidizing Bacteria in the β Subdivision of theProteobacteria
Authors: Nold Stephen C. , Zhou Jizhong , Devol Allan H. , Tiedje James M. 
Published: July 2002

ABSTRACTThe diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while β-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that γ-proteobacteria such asNitrosococcus oceaniare minor members of marine sediment ammonia-oxidizing communities.
Molecular Characterization of Bacterial Populations in Petroleum-Contaminated Groundwater Discharged from Underground Crude Oil Storage Cavities
Authors: Watanabe Kazuya , Watanabe Kanako , Kodama Yumiko , Syutsubo Kazuaki , Harayama Shigeaki 
Published: July 2002

ABSTRACT

Petroleum-contaminated groundwater discharged from underground crude oil storage cavities (cavity groundwater) harbored more than 10
6
microorganisms ml
−1
, a density 100 times higher than the densities in groundwater around the cavities (control groundwater). To characterize bacterial populations growing in the cavity groundwater, 46 PCR-amplified almost full-length 16S ribosomal DNA (rDNA) fragments were cloned and sequenced, and 28 different sequences were obtained. All of the sequences were affiliated with the
Proteobacteria
; 25 sequences (43 clones) were affiliated with the epsilon subclass, 2 were affiliated with the beta subclass, and 1 was affiliated with the delta subclass. Two major clusters (designated clusters 1 and 2) were found for the epsilon subclass proteobacterial clones; cluster 1 (25 clones) was most closely related to
Thiomicrospira denitrificans
(88% identical in nucleotide sequence), while cluster 2 (11 clones) was closely related to
Arcobacter
spp. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rDNA fragments showed that one band was detected most strongly in cavity groundwater profiles independent of storage oil type and season. The sequence of this major band was identical to the sequences of most of the cluster 1 clones. Fluorescence in situ hybridization (FISH) indicated that the cluster 1 population accounted for 12 to 24% of the total bacterial population. This phylotype was not detected in the control groundwater by DGGE and FISH analyses. These results indicate that the novel members of the epsilon subclass of the
Proteobacteria
grow as major populations in the petroleum-contaminated cavity groundwater.
Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR
Authors: Mesarch Matthew B. , Nakatsu Cindy H. , Nies Loring 
Published: July 2002

ABSTRACT

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10
2
to 10
3
gene copies, which was lowered to 10
0
to 10
1
gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.
Diversity of
Salmonella
Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions
Authors: Baudart Julia , Lemarchand Karine , Brisabois Anne , Lebaron Philippe 
Published: July 2002

ABSTRACT

Salmonella
species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater.
Salmonella
spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of
Salmonella
strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of
Salmonella
isolates.
Construction and Evaluation of a Novel Bifunctional
N
-Carbamylase–
d
-Hydantoinase Fusion Enzyme
Authors: Kim Geun-Joong , Lee Dong-Eun , Kim Hak-Sung 
Published: July 2002

ABSTRACT

A fully enzymatic process employing two sequential enzymes,
d
-hydantoinase and
N
-carbamylase, is a typical case requiring combined enzyme activity for the production of
d
-amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the
d
-hydantoinase. Firstly, maltose-binding protein (MBP) gene of
E. coli
was fused with
d
-hydantoinase gene from
Bacillus stearothermophilus
SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native
d
-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of
d
-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of
N
-carbamylase (304 amino acids [aa]) from
Agrobacterim radiobacter
NRRL B11291 and
d
-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the
d
-amino acids. A related
d
-hydantoinase (HYD1) gene from
Bacillus thermocatenulatus
GH2 was also fused with the
N
-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.
trans-o
-Hydroxybenzylidenepyruvate Hydratase-Aldolase as a Biocatalyst
Authors: Eaton Richard W. 
Published: July 2002

ABSTRACT

The hydratase-aldolase-catalyzed conversion of
trans-o
-hydroxybenzylidenepyruvate to salicylaldehyde and pyruvate is an intermediate reaction in the conversion of naphthalene to salicylate by bacteria. Here, a variety of aromatic aldehydes and some nonaromatic aldehydes together with pyruvate have been shown to be substrates for aldol condensations catalyzed by this enzyme in extracts of the recombinant strain
Escherichia coli
JM109(pRE701). Some of the products of these reactions were also compared as substrates in the opposite (hydration-aldol cleavage) reaction.
Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation
Authors: Baty Ace M. , Eastburn Callie C. , Diwu Zhenjun , Techkarnjanaruk Somkiet , Goodman Amanda E. , Geesey Gill G. 
Published: July 2002

ABSTRACT

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium
Pseudoalteromonas
sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the
chiA
promoter was used to evaluate
chiA
gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97–
N
-acetyl-β-
d
-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of
chiA
expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.
Real-Time Measurements of the Interaction between Single Cells of
Listeria monocytogenes
and Nisin on a Solid Surface
Authors: Budde Birgitte Bjørn , Jakobsen Mogens 
Published: July 2002

ABSTRACT

A method to obtain real-time measurements of the interactions between nisin and single cells of
Listeria monocytogenes
on a solid surface was developed. This method was based on fluorescence ratio-imaging microscopy and measurements of changes in the intracellular pH (pH
i
) of carboxyfluorescein succinimidyl ester-stained cells during exposure to nisin. Immobilized cells were placed in a chamber mounted on a microscope and attached to a high-precision peristaltic pump which allowed rapid changes in the nisin concentration. In the absence of nisin, the pH
i
of
L. monocytogenes
was almost constant (approximately pH 8.0) and independent of the external pH in the pH range from 5.0 to 9.0. In the presence of nisin, dissipation of the pH gradient (ΔpH) was observed, and this dissipation was both time and nisin concentration dependent. The dissipation of ΔpH resulted in cell death, as determined by the number of CFU. In the model system which we used the immobilized cells were significantly more resistant to nisin than the planktonic cells. The kinetics of ΔpH dissipation for single cells revealed a variable lag phase depending on the nisin concentration, which was followed by a very rapid decrease in pH
i
within 1 to 2 min. The differences in nisin sensitivity between single cells in a
L. monocytogenes
population were insignificant for cells grown to the stationary phase in a liquid laboratory substrate, but differences were observed for cells grown on an agar medium under similar conditions, which resulted in some cells having increased resistance to nisin.
Reduction of Technetium(VII) by
Desulfovibrio fructosovorans
Is Mediated by the Nickel-Iron Hydrogenase
Authors: De Luca Gilles , de Philip Pascale , Dermoun Zorah , Rousset Marc , Verméglio André 
Published: July 2002

ABSTRACT

Resting cells of the sulfate-reducing bacterium
Desulfovibrio fructosovorans
grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase. The role of cytochrome
c
3
in the mechanism is also discussed.
Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic Bacterium
Photorhabdus luminescens
Authors: Bowen David J. , Ensign Jerald C. 
Published: July 2002

ABSTRACT

Cells of the entomopathogenic bacterium
Photorhabdus luminescens
contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed to
Galleria mellonella
larvae.
DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in
Bradyrhizobium elkanii
Authors: Yasuta Tsuyoshi , Okazaki Shin , Mitsui Hisayuki , Yuhashi Ken-Ichi , Ezura Hiroshi , Minamisawa Kiwamu 
Published: July 2002

ABSTRACT

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by
Bradyrhizobium elkanii
. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing
rtxA
showed that several open reading frames (ORFs) were located downstream of
rtxA
. A large-deletion mutant of
B. elkanii
, USDA94Δ
rtx
::Ω1, which lacks
rtxA
, ORF1 (
rtxC
), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains
rtxA
and these ORFs, complemented rhizobitoxine production in USDA94Δ
rtx
::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least
rtxA
and
rtxC
are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of
rtxA
indicated that
rtxA
is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of
rtxC
produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the
rtxC
product was highly homologous to the fatty acid desaturase of
Pseudomonas syringae
and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that
rtxC
encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.
Poly(3-Hydroxybutyrate) Synthesis Genes in
Azotobacter
sp. Strain FA8
Authors: Pettinari M. Julia , Vázquez Gustavo J. , Silberschmidt Daniel , Rehm Bernd , Steinbüchel Alexander , Méndez Beatriz S. 
Published: July 2002

ABSTRACT

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in
Azotobacter
sp. FA8 were cloned and analyzed. A PHB polymerase gene (
phbC
) was found downstream from genes coding for β-ketothiolase (
phbA
) and acetoacetyl-coenzyme A reductase (
phbB
). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The
phbC
gene from this strain was introduced into
Ralstonia eutropha
PHB-4 (
phbC
-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.
Microbial Thiocyanate Utilization under Highly Alkaline Conditions
Authors: Sorokin Dimitry Y. , Tourova Tatyana P. , Lysenko Anatoly M. , Kuenen J. Gijs 
Published: July 2002

ABSTRACT

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS
−
) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division
Proteobacteria
(the
Halomonas
group for the heterotrophs and the genus
Thioalkalivibrio
for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus
Thioalkalivibrio
. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus
Thioalkalivibrio
according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO
−
) to ammonia and CO
2
. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.