bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts
Authors: Szeto Tim H. , Rowland Susan L. , Rothfield Lawrence I. , King Glenn F.
Published: November 2002

MinD is a widely conserved ATPase that has been demonstrated to play a pivotal role in selection of the division site in eubacteria and chloroplasts. It is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif. MinD localizes to the cytoplasmic face of the inner membrane in
Escherichia coli
, and its association with the inner membrane is a prerequisite for membrane recruitment of the septation inhibitor MinC. However, the mechanism by which MinD associates with the membrane has proved enigmatic; it seems to lack a transmembrane domain and the amino acid sequence is devoid of hydrophobic tracts that might predispose the protein to interaction with lipids. In this study, we show that the extreme C-terminal region of MinD contains a highly conserved 8- to 12-residue sequence motif that is essential for membrane localization of the protein. We provide evidence that this motif forms an amphipathic helix that most likely mediates a direct interaction between MinD and membrane phospholipids. A model is proposed whereby the membrane-targeting motif mediates the rapid cycles of membrane attachment–release–reattachment that are presumed to occur during pole-to-pole oscillation of MinD in
E. coli
.
The substrate binding domain of DnaK facilitates slow protein refolding
Authors: Tanaka Naoki , Nakao Shota , Wadai Hiromasa , Ikeda Shoichi , Chatellier Jean , Kunugi Shigeru
Published: November 2002

We examined the effects of a fragment of the substrate binding domain of DnaK on protein refolding from chemically denatured states. The fragment DnaK384-638, containing a full-length substrate binding domain, tightly binds to the unfolded protein in solution. The effects of DnaK384-638 on the reactivation of β-galactosidase and luciferase were examined at low substrate concentration and low temperature, conditions in which the folding is significantly slow (several days) but the reactivation yield is higher than those in ordinary refolding conditions. In the presence of DnaK384-638, the maximum yield of active β-galactosidase was improved from 45% to 65% after a 48-h refolding reaction. Spectroscopic experiments showed that DnaK384-638 bound to partially structured monomers of β-galactosidase and consequently suppressed aggregation. DnaK384-638 accelerated the refolding of luciferase to attain equilibrium in 8 h. On the other hand, DnaK386-561, which has no affinity for the substrate, had no chaperone activity for the reactivation of these proteins. These results indicate that the substrate binding of DnaK384-638 facilitates slow protein refolding.
Defining thyrotropin-dependent and -independent steps of thyroid hormone synthesis by using thyrotropin receptor-null mice
Authors: Marians R. C. , Ng L. , Blair H. C. , Unger P. , Graves P. N. , Davies T. F.
Published: November 2002

The thyrotropin (TSH) receptor (TSHR) is a member of the heterotrimeric G protein-coupled family of receptors whose main function is to regulate thyroid cell proliferation as well as thyroid hormone synthesis and release. In this study, we generated a TSHR knockout (TSHR-KO) mouse by homologous recombination for use as a model to study TSHR function. TSHR-KO mice presented with developmental and growth delays and were profoundly hypothyroid, with no detectable thyroid hormone and elevated TSH. Heterozygotes were apparently unaffected. Knockout mice died within 1 week of weaning unless fed a diet supplemented with thyroid powder. Mature mice were fertile on the thyroid-supplemented diet. Thyroid glands of TSHR-KO mice produced uniodinated thyroglobulin, but the ability to concentrate and organify iodide could be restored to TSHR-KO thyroids when cultured in the presence of the adenylate cyclase agonist forskolin. Consistent with this observation was the lack of detectable sodium-iodide symporter expression in TSHR-KO thyroid glands. Hence, by using the TSHR-KO mouse, we providedin vivoevidence, demonstrating that TSHR expression was required for expression of sodium-iodide symporter but was not required for thyroglobulin expression, suggesting that the thyroid hormone synthetic pathway of the mouse could be dissociated into TSHR-dependent and -independent steps.
In vivodynamics of T cell activation, proliferation, and death in HIV-1 infection: Why are CD4+but not CD8+T cells depleted?
Authors: Ribeiro Ruy M. , Mohri Hiroshi , Ho David D. , Perelson Alan S.
Published: November 2002

Deuterated glucose labeling was used to measure thein vivoturnover of T lymphocytes. A realistic T cell kinetic model, with populations of resting and activated T cells, was fitted tod-glucose labeling data from healthy and HIV-1-infected individuals before and after antiretroviral treatment. Our analysis highlights why HIV-1 infection, which increases the fraction of both CD4+and CD8+T lymphocytes that are proliferating (Ki67+), leads to CD4 but not CD8 depletion. We find that HIV-1 infection tends to increase the rates of death and proliferation of activated CD4+T cells, and to increase the rate at which resting CD4 T cells become activated, but does not increase the fraction of activated CD4+T cells, consistent with their preferential loss in HIV-1-infected individuals. In contrast, HIV-1 infection does not lead to an increase in proliferation or death rates of activated CD8+T cells, but did increase the fraction of activated CD8+T cells, consistent with these cells remaining in an activated state longer and undergoing more rounds of proliferation than CD4+T cells. Our results also explain why telomeres shorten in CD8+cells, but not in CD4+cells of HIV-1-infected patients, compared with age-matched controls.
Linking climate change and biological invasions: Ocean warming facilitates nonindigenous species invasions
Authors: Stachowicz John J. , Terwin Jeffrey R. , Whitlatch Robert B. , Osman Richard W.
Published: November 2002

The spread of exotic species and climate change are among the most serious global environmental threats. Each independently causes considerable ecological damage, yet few data are available to assess whether changing climate might facilitate invasions by favoring introduced over native species. Here, we compare our long-term record of weekly sessile marine invertebrate recruitment with interannual variation in water temperature to assess the likely effect of climate change on the success and spread of introduced species. For the three most abundant introduced species of ascidian (sea squirt), the timing of the initiation of recruitment was strongly negatively correlated with winter water temperature, indicating that invaders arrived earlier in the season in years with warmer winters. Total recruitment of introduced species during the following summer also was positively correlated with winter water temperature. In contrast, the magnitude of native ascidian recruitment was negatively correlated with winter temperature (more recruitment in colder years) and the timing of native recruitment was unaffected. In manipulative laboratory experiments, two introduced compound ascidians grew faster than a native species, but only at temperatures near the maximum observed in summer. These data suggest that the greatest effects of climate change on biotic communities may be due to changing maximum and minimum temperatures rather than annual means. By giving introduced species an earlier start, and increasing the magnitude of their growth and recruitment relative to natives, global warming may facilitate a shift to dominance by nonnative species, accelerating the homogenization of the global biota.
Auditory looming perception in rhesus monkeys
Authors: Ghazanfar Asif A. , Neuhoff John G. , Logothetis Nikos K.
Published: November 2002

The detection of approaching objects can be crucial to the survival of an organism. The perception of looming has been studied extensively in the visual system, but remains largely unexplored in audition. Here we show a behavioral bias in rhesus monkeys orienting to “looming” sounds. As in humans, the bias occurred for harmonic tones (which can reliably indicate single sources), but not for broadband noise. These response biases to looming sounds are consistent with an evolved neural mechanism that processes approaching objects with priority.
Tissue factor deficiency causes cardiac fibrosis and left ventricular dysfunction
Authors: Pawlinski R. , Fernandes A. , Kehrle B. , Pedersen B. , Parry G. , Erlich J. , Pyo R. , Gutstein D. , Zhang J. , Castellino F. , Melis E. , Carmeliet P. , Baretton G. , Luther T. , Taubman M. , Rosen E. , Mackman N.
Published: November 2002

Exposure of blood to tissue factor (TF) activates the extrinsic (TF:FVIIa) and intrinsic (FVIIIa:FIXa) pathways of coagulation. In this study, we found that mice expressing low levels of human TF (≈1% of wild-type levels) in an mTF
−/−
background had significantly shorter lifespans than wild-type mice, in part, because of spontaneous fatal hemorrhages. All low-TF mice exhibited a selective heart defect that consisted of hemosiderin deposition and fibrosis. Direct intracardiac measurement demonstrated a 30% reduction (
P
< 0.001) in left ventricular function in 8-month-old low-TF mice compared with age-matched wild-type mice. Mice expressing low levels of murine FVII (≈1% of wild-type levels) exhibited a similar pattern of hemosiderin deposition and fibrosis in their hearts. In contrast, FIX
−/−
mice, a model of hemophilia B, had normal hearts. Cardiac fibrosis in low-TF and low-FVII mice appears to be caused by hemorrhage from cardiac vessels due to impaired hemostasis. We propose that TF expression by cardiac myocytes provides a secondary hemostatic barrier to protect the heart from hemorrhage.
SWI/SNF-dependent long-range remodeling of yeast
HIS3
chromatin
Authors: Kim Yeonjung , Clark David J.
Published: November 2002

Current models for the role of the SWI/SNF chromatin remodeling complex in gene regulation are focused on promoters, where the most obvious changes in chromatin structure occur. Here we present evidence that the SWI/SNF complex is involved in the remodeling of the chromatin structure of an entire gene
in vivo
. We compared the native chromatin structures of a small yeast plasmid containing the
HIS3
gene purified from uninduced and induced cells. Relative to uninduced chromatin, induced chromatin displayed a large reduction in negative supercoiling, a large reduction in sedimentation rate, and increased accessibility to restriction enzymes with sites located both near and far from the
HIS3
promoter. These observations indicate that the entire plasmid was remodeled as a result of induction. Loss of supercoiling required the presence of the SWI/SNF remodeling complex and the activator Gcn4p
in vivo
. The TATA boxes were not required, suggesting that remodeling was not the result of transcription. The induction-dependent loss of negative supercoiling was not apparent in cells, indicating that the supercoils were lost preferentially from induced chromatin during purification. Thus, induced
HIS3
chromatin has a highly labile structure that is revealed as a result of purification. It is concluded that induction of
HIS3
creates a domain of labile chromatin structure that extends far beyond the promoter to include the entire gene. We propose that the SWI/SNF complex is recruited to the
HIS3
promoter by Gcn4p and then directs remodeling of a chromatin domain, with important implications for transcription.
Noninvasive imaging of protein–protein interactions in living subjects by using reporter protein complementation and reconstitution strategies
Authors: Paulmurugan R. , Umezawa Y. , Gambhir S. S.
Published: November 2002

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein—protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD–Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein–protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein–protein interactions.
Kidney-specific chloride channel, OmClC-K, predominantly expressed in the diluting segment of freshwater-adapted tilapia kidney
Authors: Miyazaki Hiroaki , Kaneko Toyoji , Uchida Shinichi , Sasaki Sei , Takei Yoshio
Published: November 2002

The kidney plays an important role in osmoregulation in freshwater teleosts, which are exposed to the danger of osmotic loss of Na
+
and Cl
−
. However, ion-transport mechanisms in the kidney are poorly understood, and ion transporters of the fish nephron have not been identified thus far. From Mozambique tilapia,
Oreochromis mossambicus
, we have cloned a chloride channel, which is a homologue of the mammalian kidney-specific chloride channel, ClC-K. The cDNA of the channel, named OmClC-K, encodes a protein whose amino acid sequence has high homology to
Xenopus
and mammalian ClC-K (
Xenopus
ClC-K, 41.8%; rat ClC-K2, 40.9%; rat ClC-K1, 40.1%). The mRNA of OmClC-K was expressed exclusively in the kidney, and the expression level of mRNA was increased more in freshwater-adapted fish than seawater-adapted fish. The immunohistochemical study using a specific antibody showed that OmClC-K-positive cells were specifically located in the distal nephron segments. Immunoelectron microscopy further showed that immunoreaction of OmClC-K was recognizable on the structure of basolateral membrane infoldings in the distal tubule cells. The localization of OmClC-K and its induction in hypoosmotic media suggest that OmClC-K is involved in Cl
−
reabsorption in the distal tubule of freshwater-adapted tilapia.
Altered host plant volatiles are proxies for sex pheromones in the gall wasp
Antistrophus rufus
Authors: Tooker John F. , Koenig Wilfried A. , Hanks Lawrence M.
Published: November 2002

We describe a previously uncharacterized function for changes in plant chemistry induced by phytophagous insects: to provide cues for mate location. Larvae of the gall wasp
Antistrophus rufus
Gillette (Hymenoptera: Cynipidae) feed within inconspicuous galls inside the flowering stems of the prairie perennials
Silphium laciniatum
L. and
Silphium terebinthinaceum
Jacquin (Asteraceae). Adult male
A. rufus
emerge before females and are challenged with locating mates that are sequestered within dead plant stems that occur in a matrix of dead vegetation. Allozyme studies revealed complete reproductive isolation between wasp subpopulations in the two plant species. In laboratory bioassays, males responded only to their natal plant species, antennating the stem surface. Males from
S. laciniatum
also responded to hexane extracts of
S. laciniatum
stems, and extracts contained much higher concentrations of monoterpenes (α-pinene, β-pinene, and camphene) than did
S. terebinthinaceum
. Ratios of “+” and “−” enantiomers of α- and β-pinene approximated 50:50 for nongalled
S. laciniatum
stems but strongly differed from 50:50 in galled stems, with “+” and “−” enantiomers strongly dominant in different plants. In bioassays, male wasps from
S. laciniatum
responded to a synthetic blend of the monoterpenes in enantiomeric ratios characteristic of galled stems. Male
A. rufus
rely entirely on olfaction to locate females within stems in a complex prairie habitat, and gall wasps themselves apparently influence the plant to modify ratios of monoterpene enantiomers. These plant volatiles serve as a signal for males, acting as a sex pheromone proxy for females concealed within plant tissues.
Escherichia coli
RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination
Authors: Kantake Noriko , Madiraju Murty V. V. M. , Sugiyama Tomohiko , Kowalczykowski Stephen C.
Published: November 2002

We present biochemical evidence for the functional similarity of
Escherichia coli
RecO protein and bacteriophage T4 UvsY protein to eukaryotic Rad52 protein. Although Rad52 protein is conserved in eukaryotes, no sequence homologue has been found in prokaryotes or archeabacteria. Rad52 protein has two unique activities: facilitation of replication protein-A (RPA) displacement by Rad51 protein and annealing of RPA–single-stranded DNA (ssDNA) complexes. Both activities require species-specific interaction between Rad52 protein and RPA. Both RecO and UvsY proteins also possess the former property with regard to their cognate ssDNA-binding protein. Here, we report that RecO protein anneals ssDNA that is complexed with only its cognate ssDNA-binding protein, suggesting the involvement of species-specific interactions. Optimal activity for RecO protein occurs after formation of a 1:1 complex with SSB protein. RecR protein, which is known to stimulate RecO protein to facilitate SSB protein displacement by RecA protein, inhibits annealing by RecO protein, suggesting that RecR protein may regulate the choice between the DNA strand invasion versus annealing pathways. In addition, we show that UvsY protein anneals ssDNA; furthermore, ssDNA, which is complexed only with its cognate ssDNA-binding protein, is annealed in the presence of UvsY protein. These results indicate that RecO and possibly UvsY proteins are functional counterparts of Rad52 protein. Based on the conservation of these functions, we propose a modified double-strand break repair model that includes DNA annealing as an important intermediate step.
NMR investigation of Fischer-Tropsch waxes. II. Hard wax
Authors: E C Reynhardt
Published: November 2002

For pt.I see J.A.J. Lourens and E.C. Reynhardt, ibid., vol.12, p.1963 (1970). The proton spin-lattice relaxation time and the liquid content of polycrystalline hard wax, average chain length m=50 carbon atoms and chain length disorder Delta m=15.7, were determined as a function of temperature (385>T>118K). In the low-temperature phase (T<308K) the activation energy and pre-exponential factor of threefold reorientations of methyl groups are 7.9+or-0.4 kJ mole-1and 1.46*10-12s respectively. All methyl groups have the same correlation time. The motional parameters associated with this motion in waxes are independent of chain length or chain length disorder. In the high-temperature region of the low-temperature phase, T1is dominated by reorientational-translational (RET) motions. The activation energy is 21.3+or-0.8 kJ mole-1. A linear relationship exists between ( Delta m/m)1/2and the activation energies for RET motions in waxes. The high-temperature phase (T>308K) is dominated by the gradual melting of the crystalline phase of the wax. Linear relationships exist between the percentage liquid in the wax and the specific volume and thermal conductivity of the wax. The thermogram of the wax is discussed in terms of the melting process of the wax and the effect of this process on the average chain length and chain length disorder.
Rhodanobactersp. Strain BPC1 in a Benzo[a]pyrene-Mineralizing Bacterial Consortium
Authors: Kanaly Robert A. , Harayama Shigeaki , Watanabe Kazuya
Published: November 2002

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol.66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genusRhodanobacter, whose type strain,Rhodanobacter lindaniclasticusRP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.
Purification and Characterization of Carbazole 1,9a-Dioxygenase, a Three-Component Dioxygenase System of
Pseudomonas resinovorans
Strain CA10
Authors: Nam Jeong-Won , Nojiri Hideaki , Noguchi Haruko , Uchimura Hiromasa , Yoshida Takako , Habe Hiroshi , Yamane Hisakazu , Omori Toshio
Published: November 2002

The carbazole 1,9a-dioxygenase (CARDO) system of
Pseudomonas resinovorans
strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in
Escherichia coli
strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the
k
cat
/

K
m

for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.
Effects of Dietary Fat Source and Subtherapeutic Levels of Antibiotic on the Bacterial Community in the Ileum of Broiler Chickens at Various Ages
Authors: Knarreborg Ane , Simon Mary Alice , Engberg Ricarda M. , Jensen Bent Borg , Tannock Gerald W.
Published: November 2002

The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed
−1
and salinomycin at 40 mg kg of feed
−1
) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and
Clostridium perfringens
with age of the chickens was demonstrated. Lactobacilli and
C. perfringens
were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of
C. perfringens
type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.
Construction and Evaluation of Plasmid Vectors Optimized for Constitutive and Regulated Gene Expression in
Burkholderia cepacia
Complex Isolates
Authors: Lefebre Matthew D. , Valvano Miguel A.
Published: November 2002

Genetic studies with
Burkholderia cepacia
complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in
Escherichia coli
are inefficient in
B. cepacia
. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from
Bordetella bronchiseptica
, to construct a set of vectors useful for gene expression in
B. cepacia
. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from
Burkholderia
sp. strain LB400 or the arabinose-inducible
P
BAD
promoter from
E. coli
. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-
gfp
) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the
B. cepacia
complex. We also demonstrate that
B. cepacia
carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of
Acanthamoeba polyphaga
.
Cloning and Characterization of
lin
Genes Responsible for the Degradation of Hexachlorocyclohexane Isomers by
Sphingomonas paucimobilis
Strain B90
Authors: Kumari Rekha , Subudhi Sanjukta , Suar Mrutyunjay , Dhingra Gauri , Raina Vishakha , Dogra Charu , Lal Sukanya , van der Meer Jan Roelof , Holliger Christof , Lal Rup
Published: November 2002

Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, α, β, γ, and δ. While all these isomers pose serious environmental problems, β-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by
Sphingomonas paucimobilis
strain B90 and characterized the
lin
genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the
linA
gene encoding HCH dehydrochlorinase, which were designated
linA1
and
linA2
, were found in
S. paucimobilis
B90
.
The
linA1
and
linA2
genes could be expressed in
Escherichia coli
, leading to dehydrochlorination of α-, γ-, and δ-HCH but not of β-HCH, suggesting that
S. paucimobilis
B90 contains another pathway for the initial steps of β-HCH degradation. The cloning and characterization of the halidohydrolase (
linB
), dehydrogenase (
linC
and
linX
), and reductive dechlorinase (
linD
) genes from
S. paucimobilis
B90 revealed that they share ∼96 to 99% identical nucleotides with the corresponding genes of
S. paucimobilis
UT26. No evidence was found for the presence of a
linE
-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the
linA1
and
linA2
genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between
linA1
and
linA2
, which formed
linA
of strain UT26.
Benzothiazole Degradation by
Rhodococcus pyridinovorans
Strain PA: Evidence of a Catechol 1,2-Dioxygenase Activity
Authors: Haroune Nicolas , Combourieu Bruno , Besse Pascale , Sancelme Martine , Reemtsma Thorsten , Kloepfer Achim , Diab Amer , Knapp Jeremy S. , Baumberg Simon , Delort Anne-Marie
Published: November 2002

The pathway for biodegradation of benzothiazole (BT) and 2-hydroxybenzothiazole (OBT) by
Rhodococcus pyridinovorans
strain PA was studied in detail. The kinetics of biodegradation were monitored by in situ
1
H nuclear magnetic resonance (NMR) in parallel with reversed-phase high-performance liquid chromatography (HPLC). Successive oxidations from BT to OBT and then from OBT to dihydroxybenzothiazole were observed. Further insight was obtained by using a mutant strain with impaired ability to grow on BT and OBT. The precise structure of another intermediate was determined by in situ two-dimensional
1
H-
13
C NMR and HPLC-electrospray ionization mass spectrometry; this intermediate was found to be a ring-opening product (a diacid structure). Detection of this metabolite, together with the results obtained by
1
H and
19
F NMR when cells were incubated with 3-fluorocatechol, demonstrated that a catechol 1,2-dioxygenase is involved in a pathway for biodegradation of BTs in this
Rhodococcus
strain. Our results show that catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities may both be involved in the biodegradation of BTs depending on the culture conditions.
Composition and Function of Sulfate-Reducing Prokaryotes in Eutrophic and Pristine Areas of the Florida Everglades
Authors: Castro Hector , Reddy K. R. , Ogram Andrew
Published: November 2002

As a result of agricultural activities in regions adjacent to the northern boundary of the Florida Everglades, a nutrient gradient developed that resulted in physicochemical and ecological changes from the original system. Sulfate input from agricultural runoff and groundwater is present in soils of the Northern Everglades, and sulfate-reducing prokaryotes (SRP) may play an important role in biogeochemical processes such as carbon cycling. The goal of this project was to utilize culture-based and non-culture-based approaches to study differences between the composition of assemblages of SRP in eutrophic and pristine areas of the Everglades. Sulfate reduction rates and most-probable-number enumerations revealed SRP populations and activities to be greater in eutrophic zones than in more pristine soils. In eutrophic regions, methanogenesis rates were higher, the addition of acetate stimulated methanogenesis, and SRP able to utilize acetate competed to a limited degree with acetoclastic methanogens. A surprising amount of diversity within clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) genes was observed, and the majority of DSR sequences were associated with gram-positive spore-forming
Desulfotomaculum
and uncultured microorganisms. Sequences associated with
Desulfotomaculum
fall into two categories: in the eutrophic regions, 94.7% of the sequences related to
Desulfotomaculum
were associated with those able to completely oxidize substrates, and in samples from pristine regions, all
Desulfotomaculum-
like sequences were related to incomplete oxidizers. This metabolic selection may be linked to the types of substrates that
Desulfotomaculum
spp. utilize; it may be that complete oxidizers are more versatile and likelier to proliferate in nutrient-rich zones of the Everglades.
Desulfotomaculum
incomplete oxidizers may outcompete complete oxidizers for substrates such as hydrogen in pristine zones where diverse carbon sources are less available.
Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from
Lactobacillus sanfranciscensis
CB1
Authors: De Angelis Maria , Mariotti Liberato , Rossi Jone , Servili Maurizio , Fox Patrick F. , Rollán Graciela , Gobbetti Marco
Published: November 2002

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as
Lactobacillus sanfranciscensis
CB1;
Lactobacillus brevis
AM1, AM8, and 10A;
Lactobacillus hilgardii
51B; and
Lactobacillus fructivorans
DD3 and DA106 showed all three enzyme activities.
Lactobacillus plantarum
B14 did not show CK activity.
L. sanfranciscensis
CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of
Lactobacillus sakei. L. sanfranciscensis
CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in
L. sanfranciscensis
CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by
L. sanfranciscensis
CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by
L. sanfranciscensis
CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.
Growth of Iron(III)-Reducing Bacteria on Clay Minerals as the Sole Electron Acceptor and Comparison of Growth Yields on a Variety of Oxidized Iron Forms
Authors: Kostka Joel E. , Dalton Dava D. , Skelton Hayley , Dollhopf Sherry , Stucki Joseph W.
Published: November 2002

Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe. While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor. Here we have demonstrated that a pure culture of
Shewanella oneidensis
strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor. Pure cultures of
S. oneidensis
were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors [smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen] in the same minimal medium. Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon). Cell counts showed that similar growth of
S. oneidensis
(10
8
cells ml
−1
) occurred with smectitic Fe(III) and on other Fe forms [amorphous Fe(III) oxyhydroxide, and Fe citrate] or oxygen as the electron acceptor. In contrast, cell yields of
S. oneidensis
measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions. Over a range of particle loadings (0.5 to 4 g liter
−1
), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced. From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the
Bacteria
(
Clostridium
and
Desulfitobacterium
) and the δ-
Proteobacteria
(members of the
Geobacteraceae
). Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe(III) oxide minerals and is dependent upon the mineral surface area available. Iron(III) bound in clay minerals should be considered an important electron acceptor supporting the growth of bacteria in soils or sedimentary environments.
Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event
Authors: Norris Tracy B. , Wraith Jon M. , Castenholz Richard W. , McDermott Timothy R.
Published: November 2002

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65°C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the
Bacteria
or
Archaea
domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the
Acidobacterium
(51%) and
Planctomyces
(18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50°C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50°C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.
A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria
Authors: Goh Yan Y. , Ho Bow , Ding Jeak L.
Published: November 2002

Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in anEscherichia colihost. The EGFPiproteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from theE. colilysates, the binding affinities of the EGFPiG10 and G12 to lipid A were established. TheKDvalues of 7.16 × 10−7M for G10 and 8.15 × 10−8M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFPibiosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFPispecifically tagged gram-negative bacteria likeE. coliandPseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFPiin detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.
Rapid Identification, Differentiation, and Proposed New Taxonomic Classification of
Bifidobacterium lactis
Authors: Ventura Marco , Zink Ralf
Published: November 2002

Identification of
Bifidobacterium lactis
and
Bifidobacterium animalis
is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of
B. lactis
DSM10140
T
,
B. animalis
ATCC 25527
T
, and six potential
B. lactis
strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of
B. animalis
. All of the molecular techniques used (
B. lactis
or
B. animalis
PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that
B. lactis
and
B. animalis
form two main groups and suggest a revision of the strains assigned to
B. animalis
. We propose that
B. lactis
should be separated from
B. animalis
at the subspecies level.
Quantification of Bacteria Adherent to Gastrointestinal Mucosa by Real-Time PCR
Authors: Huijsdens Xander W. , Linskens Ronald K. , Mak Mariëtte , Meuwissen Stephan G. M. , Vandenbroucke-Grauls Christina M. J. E. , Savelkoul Paul H. M.
Published: November 2002

The use of real-time quantitative PCR (5′ nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of
Escherichia coli
and
Bacteroides vulgatus
. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5′ nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of
E. coli
and 9 CFU of
B. vulgatus
could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to
E. coli
and
B. vulgatus
and with eight other gut microflora species. Mucosal samples spiked with known amounts of
E. coli
or
B. vulgatus
DNA showed no PCR inhibition. We conclude that the 5′ nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.
High Oral Prevalence of
Candida krusei
in Leprosy Patients in Northern Thailand
Authors: Reichart P. A. , Samaranayake L. P. , Samaranayake Y. H. , Grote M. , Pow E. , Cheung B.
Published: November 2002

Although
Candida albicans
is the most common human yeast pathogen, other
Candida
species such as
C. krusei
are now recognized as emerging agents, especially in patients with human immunodeficiency virus (HIV) disease.
C. krusei
is inherently resistant to the widely used triazole antifungal fluconazole and poses therapeutic problems, especially in systemic candidiasis. In a surveillance study of leprosy patients (with arrested or burnt-out disease) in a leprosarium in northern Thailand, we found a rate of oral carriage of
C. krusei
(36%) significantly (
P
< 0.05) higher than that for a healthy control group (10%). Among the
Candida-
positive patients, 16 of 35 (46%) carried
C. krusei
, while
C. albicans
was the second most common isolate (12 of 35 patients; 34%). The corresponding figures for the control group were 2 of 13 (15%) and 6 of 13 (46%), respectively. Studies of the antifungal resistance of the
C. krusei
isolates from patients indicated that all except one of the isolates were resistant to fluconazole, two isolates were resistant to ketoconazole, and all isolates were sensitive to amphotericin B. Evaluation of their genetic profiles by randomly amplified polymorphic DNA analysis with three different primers and subsequent analysis of the gel profiles by computerized cluster-derived dendrograms revealed that the
C. krusei
isolates from patients belonged to 10 disparate clusters, despite the origin from a single locale. These nascent findings indicate an alarmingly high prevalence of a
Candida
species resistant to a widely used antifungal in a part of the world where HIV disease is endemic.
Evidence that Rodents Are a Reservoir of Hepatitis E Virus for Humans in Nepal
Authors: He Junkun , Innis Bruce L. , Shrestha Mrigendra P. , Clayson Edward T. , Scott Robert M. , Linthicum Kenneth J. , Musser Guy G. , Gigliotti Scott C. , Binn Leonard N. , Kuschner Robert A. , Vaughn David W.
Published: November 2002

Hepatitis E virus (HEV) is an important cause of enterically transmitted hepatitis in developing countries. Sporadic autochthonous cases of hepatitis E have been reported recently in the United States and other industrialized countries. The source of HEV infection in these cases is unknown; zoonotic transmission has been suggested. Antibodies to HEV have been detected in many animals in areas where HEV is endemic and in domestic swine and rats in the United States. There is evidence supporting HEV transmission between swine and humans. Nevertheless, HEV has not been detected in wild rodents. We tested murid rodents and house shrews trapped in Nepal's Kathmandu Valley, where hepatitis E is hyperendemic, for HEV infection. The most commonly trapped species was
Rattus rattus brunneusculus
. Serum samples from 675 animals were tested for immunoglobulin G against HEV by enzyme-linked immunosorbent assay; 78 (12%) were positive, indicating acute or past infection. Antibody prevalence was higher among
R. rattus brunneusculus
and
Bandicota bengalensis
than in
Suncus murinus.
Forty-four specimens from 78 antibody-positive animals had sufficient residual volume for detection of HEV RNA (viremia) by reverse transcription-PCR. PCR amplification detected four animals (9%; three were
R. rattus brunneusculus
and one was
B. bengalensis
) with viremia. Phylogenetic analysis of the four genome sequences (405 bp in the capsid gene) recovered showed that they were identical, most closely related to two human isolates from Nepal (95 and 96% nucleotide homology, respectively), and distinct from HEV sequences isolated elsewhere. These data prove that certain peridomestic rodents acquire HEV in the wild and suggest that cross-species transmission occurs, with rodents serving as a virus reservoir for humans.