bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of 3-Ketoacyl-Acyl Carrier Protein Reductase (
fabG
) Genes Enhances Production of Polyhydroxyalkanoate Copolymer from Glucose in Recombinant
Escherichia coli
JM109
Authors: Nomura Christopher T. , Taguchi Kazunori , Gan Zhihua , Kuwabara Kazuhiro , Tanaka Tomoyo , Takase Kazuma , Doi Yoshiharu
Published: August 2005

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (
fabG
) in recombinant
Escherichia coli
has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new
fabG
gene from
Pseudomonas
sp. 61-3 was cloned and its gene product characterized. Results indicate that the
Pseudomonas
sp. 61-3 and
E. coli
FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of
fabG
genes from either
E. coli
or
Pseudomonas
sp. 61-3 with
fabH
(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant
E. coli
. SCL-MCL PHA copolymer isolated from a recombinant
E. coli
strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.
Enhanced Reactivity of
Rhizopus oryzae
Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents
Authors: Shiraga Seizaburo , Kawakami Masayuki , Ishiguro Masaji , Ueda Mitsuyoshi
Published: August 2005

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized
Rhizopus oryzae
lipase displayed on the cell surface of
Saccharomyces cerevisiae
in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10
4
-fold higher in a hydrolysis reaction using
p
-nitrophenyl palmitate and 3.8 × 10
4
-fold higher in an esterification reaction with palmitic acid and
n
-pentanol (0.2% H
2
O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.
First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon
Pyrococcus horikoshii
: Cloning, Expression, and Characterization
Authors: Sakuraba Haruhiko , Kawakami Ryushi , Ohshima Toshihisa
Published: August 2005

The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon
Pyrococcus horikoshii
was identified in the genome database, cloned, and functionally expressed in
Escherichia coli
. The recombinant enzyme was purified to homogeneity by heat treatment at 90°C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P)
27
(the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95°C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P)
27
. The

K
m

values for NAD were determined to be 0.30 and 0.40 mM when poly(P)
27
and ATP, respectively, were used as the phosphoryl donors. The

K
m

value for ATP was 0.29 mM, and the concentration of poly(P)
27
which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg
2+
, Mn
2+
, or Ni
2+
, for its activity. The deduced amino acid sequence showed a low level of identity to those of
E. coli
ATP-dependent NAD kinase (31%) and the inorganic polyphosphate/ATP-dependent NAD kinase of
Mycobacterium tuberculosis
(29%). This is the first description of the characteristics of a poly(P)/ATP-dependent NAD kinase from a hyperthermophilic archaeon.
Shewanella oneidensis
MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms
Authors: Lies Douglas P. , Hernandez Maria E. , Kappler Andreas , Mielke Randall E. , Gralnick Jeffrey A. , Newman Dianne K.
Published: August 2005

We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system,
Shewanella oneidensis
reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by
S. oneidensis
, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in
omcB
or
mtrB
show about 50% of the wild-type levels of reduction, while a
cymA
mutant shows less than 20% of the wild-type levels of reduction and a
menF
mutant shows insignificant reduction. The Fe-bead reduction defect of the
menF
mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the
menF
gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.
Expression of Nitrite and Nitric Oxide Reductases in Free-Living and Plant-Associated
Agrobacterium tumefaciens
C58 Cells
Authors: Baek Seung-Hun , Shapleigh James P.
Published: August 2005

A number of the bacteria that form associations with plants are denitrifiers. To learn more about how the association with plants affects expression of denitrification genes, the regulation of nitrite and nitric oxide reductases was investigated in
Agrobacterium tumefaciens
. Analysis of free-living cells revealed that expression of the genes encoding nitrite and nitric oxide reductases,
nirK
and
nor
, respectively, requires low-oxygen conditions, nitric oxide, and the transcriptional regulator NnrR. Expression of
nor
was monitored in plant-associated bacteria using
nor
-
gfp
fusion expression. In root association experiments, only a small percentage of the attached cells were fluorescent, even when they were incubated under a nitrogen atmosphere. Inactivation of
nirK
had no significant effect on the ability of
A. tumefaciens
to bind to plant roots regardless of the oxygen tension, but it did decrease the occurrence of root-associated fluorescent cells. When wild-type cells containing the
gfp
fusion were infiltrated into leaves, most cells eventually became fluorescent. The same result was obtained when a
nirK
mutant was used, suggesting that nitric oxide activated
nor
expression in the endophytic bacteria. Addition of a nitric oxide synthase inhibitor to block nitric oxide generation by the plant prevented
gfp
expression in infiltrated nitrite reductase mutants, demonstrating that plant-derived nitric oxide can activate
nor
expression in infiltrated cells.
Biodesulfurization of Dibenzothiophene by Microbial Cells Coated with Magnetite Nanoparticles
Authors: Shan GuoBin , Xing JianMin , Zhang HuaiYing , Liu HuiZhou
Published: August 2005

Microbial cells of
Pseudomonas delafieldii
were coated with magnetic Fe
3
O
4
nanoparticles and then immobilized by external application of a magnetic field. Magnetic Fe
3
O
4
nanoparticles were synthesized by a coprecipitation method followed by modification with ammonium oleate. The surface-modified Fe
3
O
4
nanoparticles were monodispersed in an aqueous solution and did not precipitate in over 18 months. Using transmission electron microscopy (TEM), the average size of the magnetic particles was found to be in the range from 10 to 15 nm. TEM cross section analysis of the cells showed further that the Fe
3
O
4
nanoparticles were for the most part strongly absorbed by the surfaces of the cells and coated the cells. The coated cells had distinct superparamagnetic properties. The magnetization (δ
s
) was 8.39 emu · g
−1
. The coated cells not only had the same desulfurizing activity as free cells but could also be reused more than five times. Compared to cells immobilized on Celite, the cells coated with Fe
3
O
4
nanoparticles had greater desulfurizing activity and operational stability.
Concurrent Quantitation of Total
Campylobacter
and Total Ciprofloxacin-Resistant
Campylobacter
Loads in Rinses from Retail Raw Chicken Carcasses from 2001 to 2003 by Direct Plating at 42°C
Authors: Nannapaneni Ramakrishna , Story Robert , Wiggins Keith C. , Johnson Michael G.
Published: August 2005

This is the first report on the use of a normally lethal dose of ciprofloxacin in a
Campylobacter
agar medium to kill all ciprofloxacin-sensitive
Campylobacter
spp. but allow the selective isolation and quantitation of naturally occurring presumptive ciprofloxacin-resistant
Campylobacter
CFU in rinses from retail raw chicken carcasses (RTCC). Thermophilic-group total
Campylobacter
CFU and total ciprofloxacin-resistant
Campylobacter
CFU (irrespective of species) were concurrently quantified in rinses from RTCC by direct plating of centrifuged pellets from 10 or 50 ml out of 400-ml rinse subsamples concurrently on
Campylobacter
agar and ciprofloxacin-containing
Campylobacter
agar at 42°C (detection limit = 0.90 log
10
CFU/carcass). For 2001, 2002, and 2003, countable
Campylobacter
CFU were recovered from 85%, 96%, and 57% of RTCC, while countable ciprofloxacin-resistant
Campylobacter
CFU were recovered from 60%, 59%, and 17.5% of RTCC, respectively. Total
Campylobacter
CFU loads in RTCC rinses ranged from 0.90 to 4.52, 0.90 to 4.58, and 0.90 to 4.48 log
10
CFU/carcass in 2001, 2002, and 2003, respectively. Total ciprofloxacin-resistant
Campylobacter
CFU loads in RTCC rinses ranged from 0.90 to 4.06, 0.90 to 3.95, and 0.90 to 3.04 log
10
CFU/carcass in 2001, 2002, and 2003, respectively. Overall, total
Campylobacter
loads of 0.90 to 2.0, 2 to 3, 3 to 4, 4 to 5 log
10
CFU/carcass, respectively, were recovered from 16%, 32%, 26%, and 5% of RTCC tested over the 2-year sampling period. For the same period, total ciprofloxacin-resistant
Campylobacter
loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log
10
CFU/carcass, respectively, were recovered from 24%, 11%, 7%, and 0.2% of RTCC tested. There was a steady decline in total
Campylobacter
and total ciprofloxacin-resistant
Campylobacter
loads in RTCC rinses from 2001/2002 to 2003.
Characterization of Carbohydrate-Binding Cytochromeb562from the White-Rot FungusPhanerochaete chrysosporium
Authors: Yoshida Makoto , Igarashi Kiyohiko , Wada Masahisa , Kaneko Satoshi , Suzuki Norio , Matsumura Hirotoshi , Nakamura Nobuhumi , Ohno Hiroyuki , Samejima Masahiro
Published: August 2005

cDNA encoding a hemoprotein similar to the cytochrome domain of extracellular flavocytochrome cellobiose dehydrogenase (CDH) was cloned from the white-rot fungusPhanerochaete chrysosporium. The deduced amino acid sequence implies that there is a two-domain structure consisting of an N-terminal cytochrome domain and a C-terminal family 1 carbohydrate-binding module (CBM1) but that the flavin-containing domain of CDH is not present. The gene transcripts were observed in cultures in cellulose medium but not in cultures in glucose medium, suggesting that there is regulation by carbon catabolite repression. The gene was successfully overexpressed inPichia pastoris, and the recombinant protein was designated carbohydrate-binding cytochromeb562(CBCyt.b562). The resonance Raman spectrum suggested that the heme of CBCyt.b562is 6-coordinated in both the ferric and ferrous states. Moreover, the redox potential measured by cyclic voltammetry was similar to that of the cytochrome domain of CDH. These results suggest that the redox characteristics may be similar to those of the cytochrome domain of CDH, and so CBCyt.b562may have an electron transfer function. In a binding study with various carbohydrates, CBCyt.b562was adsorbed with high affinity on both cellulose and chitin. As far as we know, this is the first example of a CBM1 connected to a domain without apparent catalytic activity for carbohydrate; this CBM1 may play a role in localization of the redox protein on the surface of cellulose or on the fungal sheath in vivo.
Methanogen Diversity Evidenced by Molecular Characterization of Methyl Coenzyme M Reductase A (
mcrA
) Genes in Hydrothermal Sediments of the Guaymas Basin
Authors: Dhillon Ashita , Lever Mark , Lloyd Karen G. , Albert Daniel B. , Sogin Mitchell L. , Teske Andreas
Published: August 2005

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (
mcrA
) and 16S rRNA genes. Members of the
Methanomicrobiales
and
Methanosarcinales
dominated the
mcrA
and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H
2
/CO
2
- and formate-utilizing family
Methanomicrobiales
, two
mcrA
and 16S rRNA lineages were closely affiliated with cultured species of the genera
Methanoculleus
and
Methanocorpusculum
. The most frequently recovered
mcrA
PCR amplicons within the
Methanomicrobiales
did not branch with any cultured genera. Within the nutritionally versatile family
Methanosarcinales
, one 16S rRNA amplicon and most of the
mcrA
PCR amplicons were affiliated with the obligately acetate utilizing species
Methanosaeta concilii
. The
mcrA
clone libraries also included phylotypes related to the methyl-disproportionating genus
Methanococcoides
. However, two
mcrA
and two 16S rRNA lineages within the
Methanosarcinales
were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H
2
/CO
2
, formate, acetate, and methylated substrates. Phylogenetic affiliations of
mcrA
and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.
Elevated Temperature Enhances Virulence ofErwinia carotovorasubsp.carotovoraStrain EC153 to Plants and Stimulates Production of the Quorum Sensing Signal,N-Acyl Homoserine Lactone, and Extracellular Proteins
Authors: Hasegawa H. , Chatterjee A. , Cui Y. , Chatterjee A. K.
Published: August 2005

Erwinia carotovorasubsp.atroseptica,E. carotovorasubsp.betavasculorum, andE. carotovorasubsp.carotovoraproduce high levels of extracellular enzymes, such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt), and the quorum-sensing signalN-acyl-homoserine lactone (AHL) at 28°C. However, the production of these enzymes and AHL by these bacteria is severely inhibited during growth at elevated temperatures (31.2°C forE. carotovorasubsp.atrosepticaand 34.5°C forE. carotovorasubsp.betavasculorumand mostE. carotovorasubsp.carotovorastrains). At elevated temperatures these bacteria produce high levels of RsmA, an RNA binding protein that promotes RNA decay.E. carotovorasubsp.carotovorastrain EC153 is an exception in that it produces higher levels of Pel, Peh, Cel, and Prt at 34.5°C than at 28°C. EC153 also causes extensive maceration of celery petioles and Chinese cabbage leaves at 34.5°C, which correlates with a higher growth rate and higher levels of rRNA and AHL. The lack of pectinase production byE. carotovorasubsp.carotovorastrain Ecc71 at 34.5°C limits the growth of this organism in plant tissues and consequently impairs its ability to cause tissue maceration. Comparative studies withahlI(the gene encoding a putative AHL synthase),pel-1, andpeh-1transcripts documented that at 34.5°C the RNAs are more stable in EC153 than in Ecc71. Our data reveal that overall metabolic activity, AHL levels, and mRNA stability are responsible for the higher levels of extracellular protein production and the enhanced virulence of EC153 at 34.5°C compared to 28°C.
Mutation of Glutamic Acid 103 of Toluene
o
-Xylene Monooxygenase as a Means To Control the Catabolic Efficiency of a Recombinant Upper Pathway for Degradation of Methylated Aromatic Compounds
Authors: Cafaro Valeria , Notomista Eugenio , Capasso Paola , Di Donato Alberto
Published: August 2005

Toluene
o
-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of
Pseudomonas stutzeri
OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and
o
-xylene. For example, the sequential monooxygenation of
o
-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the
P. stutzeri meta
pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from
o
-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from
o
-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.
Cultivation of Mesophilic Soil Crenarchaeotes in Enrichment Cultures from Plant Roots
Authors: Simon Holly M. , Jahn Courtney E. , Bergerud Luke T. , Sliwinski Marek K. , Weimer Paul J. , Willis David K. , Goodman Robert M.
Published: August 2005

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division
Crenarchaeota
over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of
Crenarchaeota
that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.
Nonpathogenic Strains ofColletotrichum lindemuthianumTrigger Progressive Bean Defense Responses during Appressorium-Mediated Penetration
Authors: Veneault-Fourrey Claire , Laugé Richard , Langin Thierry
Published: August 2005

The fungal bean pathogenColletotrichum lindemuthianumdifferentiates appressoria in order to penetrate bean tissues. We showed that appressorium development inC. lindemuthianumcan be divided into three stages, and we obtained three nonpathogenic strains, including one strain blocked at each developmental stage. H18 was blocked at the appressorium differentiation stage; i.e., no genuine appressoria were formed. H191 was blocked at the appressorium maturation stage; i.e., appressoria exhibited a pigmentation defect and developed only partial internal turgor pressure. H290 was impaired in appressorium function; i.e., appressoria failed to penetrate into bean tissues. Furthermore, these strains could be further discriminated according to the bean defense responses that they induced. Surprisingly, appressorium maturation, but not appressorium function, was sufficient to induce most plant defense responses tested (superoxide ion production and strong induction of pathogenesis-related proteins). However, appressorium function (i.e., entry into the first host cell) was necessary for avirulence-mediated recognition of the fungus.
Novel AroA with High Tolerance to Glyphosate, Encoded by a Gene of
Pseudomonas putida
4G-1 Isolated from an Extremely Polluted Environment in China
Authors: Sun Yi-Cheng , Chen Yan-Cheng , Tian Zhe-Xian , Li Feng-Mei , Wang Xin-Yue , Zhang Jing , Xiao Zhen-Long , Lin Min , Gilmartin Niamh , Dowling David N. , Wang Yi-Ping
Published: August 2005

Glyphosate has been used globally as a safe herbicide for weed control. It inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA), which is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. A
Pseudomonas putida
strain, 4G-1, was isolated from a soil heavily contaminated by glyphosate in China. Its AroA-encoding gene (
aroA
) has been cloned, sequenced, and expressed in
Escherichia coli
. Phylogenetic analysis revealed that this AroA belongs neither to class I nor to class II AroA enzymes. When compared with
E. coli
AroA, 4G-1 AroA shows similar values for

K
m

[PEP],

K
m

[S3P], and specific enzyme activity. Moreover, 4G-1 AroA exhibits high tolerance to glyphosate, which indicates a protein with a high potential for structural and functional studies of AroA in general and its potential usage for the generation of transgenic crops resistant to the herbicide.
Optically Transparent Porous Medium for Nondestructive Studies of Microbial Biofilm Architecture and Transport Dynamics
Authors: Leis Andrew P. , Schlicher Sven , Franke Hilmar , Strathmann Martin
Published: August 2005

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.
Isolation and Characterization of
Aspergillus oryzae
Vacuolar Protein Sorting Mutants
Authors: Ohneda Mamoru , Arioka Manabu , Kitamoto Katsuhiko
Published: August 2005

The vacuolar protein sorting (
vps
) system in the filamentous fungus
Aspergillus oryzae
, which has unique cell polarity and the ability to secrete large amounts of proteins, was evaluated by using mutants that missort vacuolar proteins into the medium. Vacuolar carboxypeptidase Y (CPY) fused with enhanced green fluorescent protein (EGFP) was used as a vacuolar marker. Twenty
dfc
(
d
im EGFP
f
luorescence in
c
onidia) mutants with reduced intracellular EGFP fluorescence in conidia were isolated by fluorescence-activated cell sorting from approximately 20,000 UV-treated conidia. Similarly, 22
hfm
(
h
yper-EGFP
f
luorescence released into the
m
edium) mutants with increased extracellular EGFP fluorescence were isolated by using a fluorescence microplate reader from approximately 20,000 UV-treated conidia. The
dfc
and
hfm
mutant phenotypes were pH dependent, and missorting of CPY-EGFP could vary by 10- to 40-fold depending on the ambient pH. At pH 5.5, the dfc-14 and hfm-4 mutants had an abnormal hyphal morphology that is consistent with fragmentation of vacuoles and defects in cell polarity. In contrast, the hyphal and vacuolar morphology of the dfc-14 and hfm-4 mutants was normal at pH 8.0, although CPY-EGFP accumulated in perivacuolar dot-like structures similar to the class E compartments in
Saccharomyces cerevisiae vps
mutants. In hfm-21, CPY-EGFP localized at the Spitzenkörper when the mutant was grown at pH 8.0 but not in vacuoles, suggesting that hfm-21 may transport CPY-EGFP via a novel pathway that involves the Spitzenkörper. Correlations between vacuolar protein sorting, pH response, and cell polarity are reported for the first time for filamentous fungi.
Fluorophore-Labeled Primers Improve the Sensitivity, Versatility, and Normalization of Denaturing Gradient Gel Electrophoresis
Authors: Neufeld Josh D. , Mohn William W.
Published: August 2005

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology. We tested the effect of fluorophore-labeled primers on DGGE band migration, sensitivity, and normalization. The fluorophores Cy5 and Cy3 did not visibly alter DGGE fingerprints; however, 6-carboxyfluorescein retarded band migration. Fluorophore modification improved the sensitivity of DGGE fingerprint detection and facilitated normalization of samples from multiple gels by the application of intralane standards.
Identification of Acyl-Homoserine Lactone Signal Molecules Produced by
Nitrosomonas europaea
Strain Schmidt
Authors: Burton E. O. , Read H. W. , Pellitteri M. C. , Hickey W. J.
Published: August 2005

Nitrosomonas europaea
strain Schmidt produces at least three acyl homoserine lactone (AHL) signal molecules: C
6
-homoserine lactone (HSL), C
8
-HSL, and C
10
-HSL. These compounds were identified in extracts of chemostat culture effluent by three independent methods. The concentrations of AHL in effluent were low (0.4 to 2.2 nM) but within the range known to induce AHL-responsive systems. The absence of LuxI and LuxM homologs from the genome of
N. europaea
strain Schmidt suggested that AHL synthesis occurs by an alternate pathway, possibly mediated by an HdtS homolog. To the best of our knowledge, the present report is the first to document the types and levels of AHLs produced by
N. europaea
.
Transcriptome-Based Identification of the
Sinorhizobium meliloti
NodD1 Regulon
Authors: Capela Delphine , Carrere Sébastien , Batut Jacques
Published: August 2005

The NodD1 regulon of
Sinorhizobium meliloti
was determined through the analysis of the
S. meliloti
transcriptome in response to the plant flavone luteolin and the overexpression of
nodD1
. Nine new genes regulated by both NodD1 and luteolin were identified, demonstrating that NodD1 controls few functions behind nodulation in
S. meliloti
.
Development of a Plasmid-Mediated Reporter System for In Vivo Monitoring of Gene Expression in the Archaeon
Methanosarcina acetivorans
Authors: Apolinario Ethel E. , Jackson Karin M. , Sowers Kevin R.
Published: August 2005

A plasmid-based gene reporter system has been developed to construct
lacZ
gene fusions for monitoring intrinsic promoter expression in
Methanosarcina acetivorans
. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to
lacZ
revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal
Archaea
by providing a rapid method for monitoring and quantifying gene expression.
Genetic Analysis of Two Bile Salt Hydrolase Activities in
Lactobacillus acidophilus
NCFM
Authors: McAuliffe Olivia , Cano Raul J. , Klaenhammer Todd R.
Published: August 2005

Two genes,
bshA
and
bshB
, encoding bile salt hydrolase enzymes (EC 3.5.1.24) were identified in the genome sequence of
Lactobacillus acidophilus
NCFM. Targeted inactivation of these genes via chromosomal insertion of an integration vector demonstrated different substrate specificities for these two enzymes.
Distribution of the
ermG
Gene among Bacterial Isolates from Porcine Intestinal Contents
Authors: Wang Yanping , Wang Gui-Rong , Shoemaker Nadja B. , Whitehead Terence R. , Salyers Abigail A.
Published: August 2005

The
ermG
gene was first found in the soil bacterium
Bacillus sphaericus
. More recently, it was found in several human intestinal
Bacteroides
species. We report here the first finding of
ermG
genes in gram-positive bacteria isolated from porcine feces and from under-barn manure pits used to store porcine wastes. The porcine
ermG
sequences were identical to the sequence of the
B. sphaericus ermG
gene except that six of the seven
ermG
-containing strains contained an insertion sequence element insertion in the C-terminal end of the gene. The porcine
ermG
genes were found in three different gram-positive genera, an indication that it is possible that the gene is being spread by horizontal gene transfer. A segment of a
Bacteroides
conjugative transposon that carries an
ermG
gene cross-hybridized with DNA from six of the seven porcine isolates, but the restriction patterns in the porcine strains were different from that of the
Bacteroides
conjugative transposon.
Identification of Genes Involved in Cytochrome
c
Biogenesis in
Shewanella oneidensis
, Using a Modified
mariner
Transposon
Authors: Bouhenni R. , Gehrke A. , Saffarini D.
Published: August 2005

A modified
mariner
transposon, mini
Himar RB1
, was generated to mutagenize cells of the metal-reducing bacterium
Shewanella oneidensis
. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome
c
maturation genes
ccmC
and
ccmF1
, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome
c
production.
Farnesol Concentrations Required To Block Germ Tube Formation in
Candida albicans
in the Presence and Absence of Serum
Authors: Mosel Daniel D. , Dumitru Raluca , Hornby Jacob M. , Atkin Audrey L. , Nickerson Kenneth W.
Published: August 2005

Concentrations of (
E
,
E
)-farnesol needed to inhibit germ tube formation were determined for
Candida albicans
strains A72 and SC5314 by using six different conditions known to trigger germination. For defined media, 1 to 2 μM farnesol was sufficient. However, with serum at 2 to 20%, up to 250 μM farnesol was required. Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.
Molecular Serotyping ofEscherichia coliO26:H11
Authors: Durso Lisa M. , Bono James L. , Keen James E.
Published: August 2005

Serotyping is the foundation of pathogenicEscherichia colidiagnostics; however, few laboratories have this capacity. We developed a molecular serotyping protocol that targets, genetically, the same somatic and flagellar antigens ofE. coliO26:H11 used in traditional serotyping. It correctly serotypes strains untypeable by traditional methods, affording primary laboratories serotyping capabilities.
Prevalence and Clinical Significance of Immunoglobulin A Antibodies against Tissue Transglutaminase in Patients with Diverse Chronic Liver Diseases
Authors: Germenis Anastasios E. , Yiannaki Efthalia E. , Zachou Kalliopi , Roka Violeta , Barbanis Sotirios , Liaskos Christos , Adam Kalliopi , Kapsoritakis Andreas N. , Potamianos Spyros , Dalekos Georgios N.
Published: August 2005

The prevalence of celiac disease (CD) and the prevalence and clinical significance of anti-tissue transglutaminase (tTG) antibodies (tTGAbs) in a large series of patients with chronic liver diseases were assessed. We studied 738 patients (462 with chronic viral hepatitis, 117 with autoimmune liver diseases, 113 with alcoholic or nonalcoholic fatty liver disease, and 46 with other liver disorders) and 1,350 healthy controls (HC). Immunoglobulin A (IgA) tTGAbs were measured by enzyme-linked immunosorbent assay and a microsphere-based flow cytometric assay. Positive sera were investigated for IgA antiendomysial antibodies (EmA). IgA tTGAb-positive subjects were invited to undergo a small-intestinal biopsy and HLA-DQ allele typing. Four of 1,350 HC (0.3%) tested tTGAb
+
EmA
+
and underwent a biopsy (CD confirmation in all). Four of 738 liver disease patients tested tTGAbs
+
EmA
+
(0.54%; not statistically significant). Two were HCV infected (1.24%; not statistically significant), and two had transaminasemia of unknown origin. Forty-three patients tested tTGAbs
+
EmA
−
(5.8%;
P
< 0.001 compared to HC). Inhibition experiments verified the existence of specific IgA anti-tTG reactivity. Twenty-six of 43 patients underwent a biopsy (all negative for CD). Binary logistic regression analysis revealed age (
P
= 0.008), cirrhosis (
P
= 0.004), alkaline phosphatase (
P
= 0.026), and antinuclear antibodies (
P
= 0.012) as independent risk factors for tTGAb reactivity among the patients. It was concluded that CD prevalence is the same in HC and patients with chronic liver diseases. The prevalence of tTGAbs is higher in hepatic patients compared to HC, but their specificity for CD diagnosis in this group of patients is low. tTGAbs in patients appear to be associated with the presence of autoimmunity, cirrhosis, and cholestasis, irrespective of the origin of the liver disease.
Optimization and Validation of a Multiplexed Luminex Assay To Quantify Antibodies to Neutralizing Epitopes on Human Papillomaviruses 6, 11, 16, and 18
Authors: Dias Dennis , Van Doren Jeff , Schlottmann Sonela , Kelly Sheri , Puchalski Derek , Ruiz Wanda , Boerckel Patricia , Kessler Joseph , Antonello Joseph M. , Green Tina , Brown Martha , Smith Judith , Chirmule Narendra , Barr Eliav , Jansen Kathrin U. , Esser Mark T.
Published: August 2005

A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108-115, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.
Lymphocyte Proliferation Responses Induced to Broadly Reactive Th Peptides Did Not Protect against Equine Infectious Anemia Virus Challenge
Authors: Fraser Darrilyn G. , Leib Steve R. , Zhang Bao Shan , Mealey Robert H. , Brown Wendy C. , McGuire Travis C.
Published: August 2005

The effect of immunization with five lipopeptides, three containing T-helper (Th) epitopes and two with both Th and cytotoxic T-lymphocyte (CTL) epitopes, on equine infectious anemia virus (EIAV) challenge was evaluated. Peripheral blood mononuclear cells from EIAV lipopeptide-immunized horses had significant proliferative responses to Th peptides compared with those preimmunization, and the responses were attributed to significant responses to peptides Gag from positions 221 to 245 (Gag 221-245), Gag 250-269, and Pol 326-347; however, there were no consistent CTL responses. The significant proliferative responses in the EIAV lipopeptide-immunized horses allowed testing of the hypothesis that Th responses to immunization would enhance Th and CTL responses following EIAV challenge and lessen the viral load and the severity of clinical disease. The EIAV lipopeptide-immunized group did have a significant increase in proliferative responses to Th peptides 1 week after virus challenge, whereas the control group did not. Two weeks after challenge, a significant CTL response to virus-infected cell targets occurred in the EIAV lipopeptide-immunized group compared to that in the control group. These Th and CTL responses did not significantly alter either the number of viral RNA copies/ml or disease severity. Thus, lipopeptide-induced proliferative responses and enhanced Th and CTL responses early after virus challenge were unable to control challenge virus load and clinical disease.
Clinical features and follow up of 302 patients with Mycobacterium kansasii pulmonary infection: a 50 year experience
Authors: Maliwan N , Zvetina J R
Published: August 2005

Aims

To analyse clinical features and treatment outcomes of patients with pulmonary Mycobacterium kansasii infection treated at Hines VA Hospital between 1952 and 1995, and followed up until 2003.

Findings

302 patients were confirmed to have M kansasii pulmonary infection; diagnosis was not made until death in 2%. The average age was 50 years old; 76% were white; all were men. Productive cough, dyspnoea, and chest pain were common; 16% were asymptomatic. Right sided, apical or subapical, thin walled cavitary infiltrate was the characteristic radiological feature. Heavy smoking, chronic obstructive pulmonary disease, alcoholism, peptic ulcer disease, coronary artery disease, prior tuberculosis, psychosis, prior pneumonia, and immunocompromising conditions were prevalent. Average follow up was 10 years and 2 months. PPD was positive in 58% of 179 tested. Two thirds of the patients required only first line drugs. Fourteen per cent required surgical intervention, none after 1977. Spontaneous resolution occurred in 1%. Aspergillosis developed in 4%. Bronchogenic carcinoma coexisted with M kansasii infection in 6% and followed it in 4%. Extrapulmonary malignancy coexisted with the infection in 4% and followed it in 6%; most involved head and neck. Eleven per cent of 224 deaths were attributed to M kansasii. Outcomes were affected by comorbidity, treatment compliance, rifampicin use, and extent of infection.

Conclusions

Prognosis of M kansasii pulmonary infection is good if diagnosed and treated early, together with control of underlying conditions. Clinicians should be aware of atypical radiological manifestations of the disease when coexisting with other pulmonary or immunocompromising conditions.
Diabetes and ethnic minorities
Authors: Oldroyd J , Banerjee M , Heald A , Cruickshank K
Published: August 2005

The global prevalence of diabetes for all age groups is estimated to be 2.8%. Type 2 diabetes accounts for at least 90% of diabetes worldwide. Diabetes incidence, prevalence, and disease progression varies by ethnic group. This review highlights unique aspects of the risk of developing diabetes, its overwhelming vascular complications, and their management mainly using data among South Asians and African-Caribbeans in the UK but also using non-UK data. It is concluded that although the origin of the ethnic differences in incidence need further clarification, many factors should be amenable to prevention and treatment in all ethnic groups worldwide.
Malaria in the post-genomics era: light at the end of the tunnel or just another train?
Authors: Gardiner D L , McCarthy J S , Trenholme K R
Published: August 2005

Malaria remains the third leading cause of death attributable to an infectious disease worldwide, with an estimated death toll of over 2 million per year, predominately in sub-Saharan Africa. The first serious attempt to eradicate this disease was unsuccessful, and 50 years later in 1998 a second programme coined “roll back malaria” was started. While this programme is at present unlikely to reach its stated aims, the completion of the genome sequencing projects on the human host, the mosquito vector, and the malaria parasite offers new hope. It is probable that the burden of disease caused by the most malignant form of the parasite Plasmodium falciparum can be, if not eliminated, then effectively suppressed within a generation through new and novel treatments aimed at all three arms of malaria control.
FAITHFUL FUNCTORS FROM CANCELLATIVE CATEGORIES TO CANCELLATIVE MONOIDS WITH AN APPLICATION TO ABUNDANT SEMIGROUPS
Authors: GOULD VICTORIA , KAMBITES MARK
Published: August 2005

We prove that any small cancellative category admits a faithful functor to a cancellative monoid. We use our result to show that any primitive ample semigroup is a full subsemigroup of a Rees matrix semigroup [Formula: see text] where M is a cancellative monoid and P is the identity matrix. On the other hand a consequence of a recent result of Steinberg is that it is undecidable whether a finite ample semigroup embeds as a full subsemigroup of an inverse semigroup.
IRREGULAR QUASIVARIETIES OF COMMUTATIVE BINARY MODES
Authors: MATCZAK K. , ROMANOWSKA A. B.
Published: August 2005

In an earlier paper the authors described the isomorphic lattices of quasivarieties of commutative quasigroup modes and of cancellative commutative binary modes. This paper extends the earlier result, and provides a description of the lattice of some irregular quasivarieties of commutative binary modes, i.e. quasivarieties that do not contain semilattices.
ENDS OF CUSP-UNIFORM GROUPS OF LOCALLY CONNECTED CONTINUA — I
Authors: GURALNIK DAN P.
Published: August 2005

Due to works by Bestvina–Mess, Swarup and Bowditch, we now have complete knowledge of how splittings of a word-hyperbolic group G as a graph of groups with finite or two-ended edge groups relate to the cut point structure of its boundary. It is central in the theory that ∂G is a locally connected continuum (a Peano space). Motivated by the structure of tight circle packings, we propose to generalize this theory to cusp-uniform groups in the sense of Tukia.

A Peano space X is cut-rigid, if X has no cut point, no points of infinite valence and no cut pairs consisting of bivalent points. We prove:

Theorem. Suppose X is a cut-rigid space admitting a cusp-uniform action by an infinite group. If X contains a minimal cut triple of bivalent points, then there exists a simplicial tree T, canonically associated with X, and a canonical simplicial action of Homeo(X) on T such that any infinite cusp-uniform group G of X acts cofinitely on T, with finite edge stabilizers.

In particular, if X is such that T is locally finite, then any cusp-uniform group G of X is virtually free.